Inhibitors of Protein Kinases

ABSTRACT

The present invention relates to inhibitors of cyclin-dependent kinases and therapeutic applications thereof. Furthermore, the invention relates to methods of preventing and/or treating any type of pain, inflammatory disorders, immunological diseases, proliferative diseases, infectious diseases, cardiovascular diseases and neurodegenerative diseases comprising the administration of an effective amount of at least one inhibitor of cyclin-dependent kinases.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a division of U.S. patent application Ser. No.12/451,041, filed on Mar. 3, 2010, which is a U.S. National Stage ofPCT/EP2008/054977, filed on Apr. 24, 2008, which claims priority ofPCT/EP2007/003603, filed on Apr. 24, 2007, each of which is incorporatedherein by reference in its entirety.

BACKGROUND OF THE INVENTION

Cyclin-dependent protein kinases (“CDKs”), constitute a family ofwell-conserved enzymes that play multiple roles within the cell, such ascell cycle regulation and transcriptional control (Science 1996, Vol.274:1643-1677; Ann. Rev. Cell Dev. Biol., 1997, 13:261-291).

Some members of the family, such as CDK1, 2, 3, 4, and 6 regulate thetransition between different phases of the cell cycle, such as theprogression from a quiescent stage in G1 (the gap between mitosis andthe onset of DNA replication for a new round of cell division) to S (theperiod of active DNA synthesis), or the progression from G2 to M phase,in which active mitosis and cell division occur. Other members of thisfamily o f proteins, including CDK7, 8, and 9 regulate key points in thetranscription cycle, whereas CDK5 plays a role in neuronal and secretorycell function.

CDK complexes are formed through association of a regulatory cyclinsubunit (e.g., cyclin A, B1, B2, D1, D2, D3, and E) and a catalytickinase subunit (e.g., cdc2 (CDK1), CDK2, CDK4, CDK5, and CDK6). As thename implies, the CDKs display an absolute dependence on the cyclinsubunit in order to phosphorylate their target substrates, and differentkinase/cyclin pairs function to regulate progression through specificportions of the cell cycle.

It is known that cell-cycle dysregulation, which is one of the cardinalcharacteristics of neoplastic cells, is closely associated with geneticalteration and deregulation of CDKs and their regulators, suggestingthat inhibitors of CDKs may be useful as therapeutics for proliferativediseases, such as cancer. Thus, small molecule inhibitors targeting CDKshave been the focus of extensive interest in cancer therapy (CurrentOpinion in Pharmacology, 2003(3): 362-370). The ability of inhibitingcell cycle progression suggests a general role for small moleculeinhibitors of CDKs as therapeutics for proliferative diseases, such ascancer. While inhibition of cell cycle-related CDKs is clearly relevantin oncology applications, this may not be the case for the inhibition ofRNA polymerase-regulating CDKs.

The serine/threonine kinase CDK5 along with its cofactor p25 (or thelonger cofactor, p35) has been linked to neurodegenerative disorders,and inhibitors of cdk5/p25 (or cdk5/p35) are therefore useful for thetreatment of neurodegenerative disorders such as Alzheimer's disease,Parkinson's disease, stroke, or Huntington's disease. Treatment of suchneurodegenerative disorders using CDK5 inhibitors is supported by thefinding that CDK5 is involved in the phosphorylation of tau protein (J.Biochem, 117, 741-749 (1995)). CDK5 also phosphorylates Dopamine andCyclic AMP-Regulated Phosphorprotein (DARPP-32) at threonine 75 and isthus indicated in having a role in dopaminergic neurotransmission(Nature, 402. 669-671 (1999)).

Cyclin-dependent kinase 5 (CDK5) is involved in regulating the state ofphosphorylation of DARPP-32. CDK5 was originally identified as ahomologue of p34^(cdc2) protein kinase. Subsequent studies have shownthat unlike cdc2, CDK5 kinase activity is not detected in dividingcells. Instead, the active form of CDK5 is present only indifferentiated neurons, where it associates with a neuron-specific 35kDa regulatory subunit, termed p35. CDK5/p35 plays a variety of roles inthe developing and adult nervous system. Recent studies have linkedmis-regulation of CDK5 to Alzheimer's disease (Kusakawa, G. et al. 2000.J. Biol. Chem. 275:17166-17172; Lee, M. S. et al. 2000. Nature405:360-364; Nath, R. et al. 2000. Biochem. Biophys. Res. Commun.274:16-21; Patrick, G. N. et al. 1999. Nature 402:615-622). In thesestudies, conversion of p35 to p25 by the action of calpain causesprolonged activation and altered localization of CDK5. In turn, cdk5/p25can hyperphosphorylate tau, disrupt cytoskeletal structure and promoteapoptosis of primary neurons.

Recent studies have also shown that CDK5 phosphorylates DARPP-32 atThr75 (Nishi, A. et al. 2000. Proc. Natl. Acad. Sci. USA 97:12840-12845;Bibb, J. A. et al. 1999. Nature 402:669-671). DARPP-32 phosphorylated atThr75 is an inhibitor of PKA (Bibb et al. 1999. Nature 402:669-671).Phosphorylation of DARPP-32 at Thr75 by CDK5, by inhibiting PKA,decreases phosphorylation of Thr34 in DARPP-32 by PKA and plays animportant modulatory role in the DARPP-32/PP1 cascade (Bibb, J. A. etal. 1999. Nature 402:669-671).

Surprisingly little is known about the regulation of CDK5 by firstmessengers and other signaling events. Therefore, there is a need in theart to provide new compounds that can be used to develop novelcompositions or drugs that can be used to treat diseases or disordersrelated to the regulation of CDK5. Furthermore, there is a need todevelop treatments for such diseases or disorders that are due, at leastin part, to an aberration or dysregulation of an intracellular signalingpathway regulated by CDK5. The present invention provides such methodsand compositions.

Furthermore, the involvement of CDK5 in pain signalling has beenreviewed in Pareek et al., PNAS 103, pp. 791-796, and in Pareek et al.,Cell Cycle 5:6, pp. 585-588. More than 50 pharmacological CDK inhibitorshave been described, some of which have potent antitumor activity(Current Opinion in Pharmacology, 2003(3): 362-370). A comprehensivereview about the known CDK inhibitors may be found in Angew. Chem. Int.Ed. Engl. 2003, 42(19):2122-2138.

The use of 2-anilino-4-phenylpyrimidine derivatives as cyclin-dependentkinase inhibitors for the treatment of e.g. cancer has been reported inWO 2005/012262. Furthermore, 2-pyridinylamino-4-thiazolyl-pyrimidinederivatives for the treatment of cancer etc. have been described in WO2005/012298. The use of 4,5-dihydro-thiazolo, oxazolo andimidazolo[4,5-h]quinazolin-8-ylamines as protein kinase inhibitors isknown from WO 2005/005438. Furthermore, indolinone derivatives andinduribin derivatives, which are useful as cyclin-dependent kinaseinhibitors have been disclosed in WO 02/081445 and WO 02/074742.Additionally, CDK inhibitors for various therapeutic applications havebeen described in WO2005/026129.

Known CDK inhibitors may be classified according to their ability toinhibit CDKs in general or according to their selectivity for a specificCDK. Flavopiridol, for example, acts as a “pan” CDK antagonist and isnot particularly selective for a specific CDK (Current Opinion inPharmacology, 2003(3): 362-370). Purine-based CDK inhibitors, such asolomoucine, roscovitine, purvanolols and CGP74514A are known to exhibita greater selectivity for CDKs 1, 2 and 5, but show no inhibitoryactivity against CDKs 4 and 6 (Current Opinion in Pharmacology, 2003(3):362-370). Furthermore, it has been demonstrated that purine-based CDKinhibitors such as roscovitine can exert anti-apoptotic effects in thenervous system (Pharmacol Ther 2002, 93:135-143) or prevent neuronaldeath in neurodegenerative diseases, such as Alzheimers's disease(Biochem Biophys Res Commun 2002 (297):1154-1158; Trends Pharmacol Sci2002 (23):417-425).

Given the tremendous potential of targeting CDKs for the therapy ofconditions such as proliferative, immunological, infectious,cardiovascular and neurodegenerative diseases, the development of smallmolecules as selective inhibitors of particular CDKs constitutes adesirable goal.

The present invention provides novel small molecule inhibitors ofcyclin-dependent kinases. Preferably, said small molecule inhibitorsshow an increased potency to inhibit a particular CDK. Said smallmolecule inhibitors may have a therapeutic utility for the treatment ofconditions such as proliferative, immunological, neurodegenerative,infectious and cardiovascular diseases. Furthermore, the small moleculeinhibitors of the present invention have surprisingly been shown toexert a beneficial effect in the treatment of inflammatory diseases andof any type of pain.

Current treatments for pain are only partially effective, and many alsocause debilitating or dangerous side effects. For example, many of thetraditional analgesics used to treat severe pain induce debilitatingside effects such as nausea, dizziness, constipation, respiratorydepression, and cognitive dysfunction (Brower, 2000).

Although there is already a broad panel of approved pain medicationslike non-narcotic analgesics, opioid analgesics, calcium channelblockers, muscle relaxants, and systemic corticosteroids available, saidtreatments remain merely empirical and, while they may relieve thesymptoms of pain, they do not lead to complete relief in most cases.This is also due to fact that the mechanisms underlying the developmentof the different types of pain are still only poorly understood.Researchers are only just beginning to appreciate the complexity anddiversity of the signaling systems used to relay nerve impulses for eachtype of pain.

Generally, pain is defined as an unpleasant sensory and emotionalexperience associated with actual or potential tissue damage, ordescribed in terms of such damage, according to the InternationalAssociation for the Study of Pain (IASP). Specifically, pain may occuras acute or chronic pain.

Acute pain occurs for brief periods of time, typically less than 1 monthand is associated with temporary disorders. It is a natural bodyresponse to let the host be aware of physiological or biochemicalalteration that could result in further damage within a short period oftime. It is felt when noxious stimuli activate high threshold mechanicaland/or thermal nociceptors in peripheral nerve endings and the evokedaction potentials in thinly myelinated (Aδ) and/or unmyelinated (C)afferent fibres reach a conscious brain. Said noxious stimuli may beprovided by injury, surgery, illness, trauma or painful medicalprocedures. Acute pain usually disappears when the underlying cause hasbeen treated or has healed. Unrelieved acute pain, however, may lead tochronic pain problems that may result in long hospital stays,rehospitalizations, visits to outpatient clinics and emergencydepartments, and increased health care costs.

In contrast to acute pain, chronic pain persists long after the initialinjury has healed and often spreads to other parts of the body, withdiverse pathological and psychiatric consequences. Chronic somatic painresults from inflammatory responses to trauma in peripheral tissues(e.g., nerve entrapment, surgical procedures, cancer, or arthritis),which leads to oversensitization of nociceptors and intense searing painresponses to normally non-noxious stimuli (hyperalgesia). Chronic painis continuous and recurrent and its intensity will vary from mild tosevere disabling pain that may significantly reduce quality of life.

Chronic pain is currently treated with conventional analgesics such asNSAIDs (Ibuprofen, Naproxen), Cox-2 inhibitors (Celecoxib, Valdecoxib,Rofecoxib) and opiates (codeine, morphin, thebain, papaverin, noscapin).For a significant number of patients however, these drugs provideinsufficient pain relief.

Another subtype of pain, inflammatory pain, can occur as acute as wellas chronic pain. Resulting injuries of tissue and neurons must not butmay develop into long-lasting chronic neuropathic pain effects insuccession to such inflammatory events.

Inflammatory pain is mediated by noxious stimuli like e.g. inflammatorymediators (e.g. cytokines, such as TNF α, prostaglandins, substance P,bradykinin, purines, histamine, and serotonine), which are releasedfollowing tissue injury, disease, or inflammation and other noxiousstimuli (e.g. thermal, mechanical, or chemical stimuli). In addition,cytokines and growth factors can influence neuronal phenotype andfunction (Besson 1999). These mediators are detected by nociceptors(sensory receptors) that are distributed throughout the periphery of thetissue. Said nociceptors are sensitive to noxious stimuli (e.g.mechanical, thermal, or chemical), which would damage tissue ifprolonged (Koltzenburg 2000). A special class of so called C-nociceptorsrepresent a class of “silent” nociceptors that do not respond to anylevel of mechanical or thermal stimuli but are activated in presence ofinflammation only.

Current approaches for the treatment of especially inflammatory pain aimat cytokine inhibition (e.g. IL1β) and suppression of pro-inflammatoryTNFα. Current approved anticytokine/antiTNFalpha treatments are based onchimeric antibodies such as Infliximab and Etanercept which reduce TNFαcirculation in the bloodstream. TNFα is one of the most importantinflammatory mediators that induces synthesis of important enzymes suchas COX-2, MMP, iNOS, cPLa₂ and others. The main drawbacks of these“biologicals”, however, reside in their immunogenic potential withattendant loss of efficacy and their kinetics that lead to a more orless digital all-or-nothing reduction of circulating TNFα. The lattercan result in severe immune suppressive side effects.

A distinct form of chronic pain, neuropathic (or neurogenic) pain,arises as a result of peripheral or central nerve dysfunction andincludes a variety of conditions that differ in etiology as well aslocation. Generally, the causes of neuropathic pain are diverse, butshare the common symptom of damage to the peripheral nerves orcomponents of central pathways. The causative factors might bemetabolic, viral or mechanical nerve lesion. Neuropathic pain isbelieved to be sustained by aberrant somatosensory processes in theperipheral nervous system, the CNS, or both. Neuropathic pain is notdirectly linked to stimulation of nociceptors, but instead, is thoughtto arise e.g. from oversensitization of glutamate receptors onpostsynaptic neurons in the gray matter (dorsal horn) of the spinalcord.

Neuropathic pain is associated with conditions such as nervedegeneration in diabetes and postherpetic neuralgia (shingles).Neuropathic pain conditions are the consequence of a number of diseasesand conditions, including diabetes, AIDS, multiple sclerosis, stump andphantom pain after amputation, cancer-related neuropathy, post-herpeticneuralgia, traumatic nerve injury, ischemic neuropathy, nervecompression, stroke, spinal cord injury.

Management of neuropathic pain remains a major clinical challenge,partly due to an inadequate understanding of the mechanisms involved inthe development and maintenance of neuropathic pain. Many existinganalgesics are ineffective in treating neuropathic pain and most ofcurrent narcotic and non-narcotic drugs do not control the pain. Currentclinical practice includes the use of a number of drug classes for themanagement of neuropathic pain, for example anticonvulsants, tricyclicantidepressants, and systemic local anaesthetics. However, the usualoutcome of such treatment is partial or unsatisfactory pain relief, andin some cases the adverse effects of these drugs outweigh their clinicalusefulness. Classic analgesics are widely believed to be poorlyeffective or ineffective in the treatment of neuropathic pain. Fewclinical studies on the use of non steroidal anti-inflammatory drugs(NSAIDs) or opiates in the treatment of neuropathic pain have beenconducted, but in those which have, the results appear to indicate thatNSAIDs are poorly effective or ineffective and opiates only work at highdoses. A review analysing the controlled clinical data for peripheralneuropathic pain (PNP) (Pain, November, 1997 73(2), 123-39) reportedthat NSAIDs were probably ineffective as analgesics for PNP and thatthere was no long-term data supporting the analgesic effectiveness ofany drug.

Available analgesic drugs often produce insufficient pain relief.Although tricyclic antidepressants and some antiepileptic drugs, forexample gabapentin, lamotrigine and carbamazepine, are efficient in somepatients, there remains a large unmet need for efficient drugs for thetreatment of these conditions.

In conclusion, there is a high unmet need for safe and effective methodsof pain treatment, in particular of chronic inflammatory and neuropathicpain.

SUMMARY OF THE INVENTION

The present invention is directed to inhibitors of cyclin-dependentkinases and to methods and compositions for treating and/or preventingany type of pain, inflammatory disorders, immunological diseases,proliferative diseases, infectious diseases, cardiovascular diseases andneurodegenerative diseases comprising: administering an effective amountof at least one inhibitor of a cyclin-dependent kinase (cdk, CDK) to asubject in need thereof. The inhibitor is selected among compoundsaccording to the general Formula I:

wherein

-   -   R¹ is —XSO₂NR⁵R⁶ or —XSO₂R⁸;    -   wherein X is a C₁₋₄ alkylene (including branched alkylene),        wherein said C₁₋₄ alkylene can be bound to R⁵ or R⁶ to form a 5-        or 6-membered heterocycle;    -   R⁵ and R⁶ independently of each other are hydrogen, C₁₋₄alkyl,        hydroxy-C₁₋₄alkyl or C₃₋₄alkenyl, C₃₋₈-cycloalkyl,        C₃₋₈-cycloalkyl-C₁₋₄alkyl or C₄₋₇-heterocycloalkyl-C₀₋₄alkyl,        C₄₋₇-aryl-C₀₋₄alkyl, C₄₋₇heteroaryl-C₀₋₄alkyl or    -   wherein R⁵ and R⁶ together with the N-atom to which they are        bound also may form a 5- to 8-membered heterocycloalkyl,    -   wherein said cycloalkyl, heterocycloalkyl, aryl, heteroaryl or        alkyl is further optionally substituted by up to 2 radicals        selected from the group consisting of halo, hydroxy,        aminocarbonyl, C₁₋₄ alkyl, hydroxy-C₁₋₄ alkyl, C₁₋₄ alkyl-O—C₁₋₄        alkyl, C₁₋₄ alkyl-O— and —NR⁵R⁶;    -   R⁸ is C₁₋₄ alkyl, hydroxy-C₂₋₄alkyl or C₃₋₄alkenyl,        C₃₋₈-cycloalkyl, C₃₋₈-cycloalkyl-C₁₋₄alkyl or        C₄₋₇-heterocycloalkyl-C₀₋₄alkyl;    -   wherein said cycloalkyl, heterocycloalkyl or alkyl is further        optionally substituted by up to 2 radicals selected from the        group consisting of halo, hydroxy, C₁₋₄ alkyl, hydroxy-C₁₋₄        alkyl, C₁₋₄ alkyl-O—C₁₋₄ alkyl, C₁₋₄ alkyl-O and —NR⁵R⁶;    -   R² is one or two substituents independently selected from        halogen and hydrogen;    -   R³ can be 1 to 3 substituents each independently selected from        the group consisting of hydrogen, halo, hydroxy, C₁₋₄alkyl,        C₃₋₇cycloalkyl, C₁₋₄ alkyl-cycloalkyl, C₁₋₄        alkyl-heterocycloalkyl, —O-heterocycloalkyl, C₁₋₄ alkoxy, C₂₋₄        alkenyloxy, —OCF₃, C₂₋₄ alkanoyl, C₁₋₄alkylsulfonyl, mono- and        di-(C₁-C₄alkyl)sulfonamido, aminocarbonyl, mono- and        di-(C₁-C₄alkyl)aminocarbonyl, aryl-C₁₋₄ alkoxy, heteroaryl-C₁₋₄        alkoxy, heterocycloalkyl-C1-4-alkoxy,        heterocycloalkyl-C1-4-alkyl, heteroaryl-C1-4-alkyl, C₁₋₄        alkyloxymethyl, hydroxy-C₁₋₄alkyloxymethyl, cyano, —COOH and        C₁-C₄ alkoxycarbonyl, wherein the above mentioned substituents        can be further substituted by radicals selected from the group        of C₁₋₄-alkyl, hydroxyl-C₀₋₄-alkyl, C₁₋₄-alkoxy, aminocarbonyl,        halo, and NR⁵R⁶;    -   R^(4a) and R^(4b) are the same or different and each is        independently hydrogen, C₁₋₄ alkyl or —NR′R″, wherein R′ and R″        are each independently hydrogen or C₁₋₄ alkyl;    -   and the N-oxide derivatives, prodrug derivatives, protected        derivatives, individual isomers and mixtures of isomers thereof;        and the pharmaceutically acceptable salts and solvates (e.g.,        hydrates) of such compounds.

As disclosed herein, the term “C₁₋₄ alkyl” is meant to include straightor branched chain saturated aliphatic hydrocarbon having 1 to 4 carbonatoms, such as methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl,isobutyl, tert-butyl.

The term “lower alkenyl” refers to straight or branched chain alkenegroups which have from 2 to 6 carbon atoms, such as, for example, vinyl,allyl, but-2-enyl, -but-3-enyl, or isopropenyl; the term “lower alkenyl”preferably represents allyl, -but-2-enyl, or -but-3-enyl.

As disclosed herein, the term “halo” is meant to include fluoro, chloro,bromo, and iodo.

The term C₃-C₈ cycloalkyl denotes the following cycloalkyls:

The term aryl denotes an aromatic mono- or bicyclic 6 to 10 memberedring system such as phenyl, naphthyl, 3-chlorophenyl, 2,6-dibromophenyl,2,4,6 tribromophenyl, 4,7-dichloronaphthyl, and preferably phenyl ornaphthyl.

The term heterocycloalkyl is meant to include a 5 to 10 membered mono-or bicyclic ring system, containing one to three heteroatomsindependently selected from oxygen, sulfur or nitrogen and is preferablyselected from the group comprising: Aziridinyl, azetidinyl,pyrrolidinyl, tetrahydrofuranyl, tetrahydrothiophenyl, piperidinyl,piperadizinyl, piperazinyl, tetrahydropyranyl, tetrahydrothiopyranyl ormorpholinyl.

The term heterocycloalkyl further comprises all heteroaryls as definedbelow, wherein all double bonds of the correspondent heteroaryls arereplaced by single bonds.

The term heteroaryl denotes a partially or fully unsaturated 5 to 10membered mono- or bicyclic ringsystem, containing one to threeheteroatoms independently selected from oxygen, sulfur or nitrogen andis preferably selected from the group consisting of:

Pyrrolyl, furanyl, thiophenyl, thienyl, imidazolyl, pyrazolyl,thiazolyl, oxazolyl, isothiazolyl, isoxazolyl, pyridinyl, pyridyl,pyrimidinyl, pyrimidyl, pyrazinyl, pyrazyl, pyradizinyl, pyradizyl,3-methylpyridyl, benzothienyl, 4-ethylbenzothienyl, 3,4-diethylfuranyl,pyrrolyl, tetrahydroquinolyl, quinolyl, tetrahydroisoquinolinyl,isoquinolinyl, benzoimidazolyl, benzothiazolyl, benzooxyzolyl,benzo[1,3]dioxolyl, indolyl, benzofuranyl, benzothiophenyl, indazolyl orchrom-2-onyl.

It is to be understood, that the term heteroaryl also comprisespartially unsaturated 5 to 10 membered mono- or bicyclic ringsystem,wherein one up to 4 double bonds of the ringsystem are replaced by asingle bond and wherein the ringsystem contains at least one doublebond.

When any variable occurs more than once in Formula I or in anysubstituent, its definition on each occurrence is independent of itsdefinition at every other occurrence. For example, when a compoundcomprises more than one R⁵ and/or R⁶ substituent, they can be the sameor different

In the compounds of formula I:

X is preferably methylene.R² is preferably hydrogen.

Preferably R⁶ is hydrogen or methyl and R⁵ is ethyl, 2-hydroxyethyl,isopropyl, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, n-propyl,t-butyl, 3-methoxy-propyl, 2-dimethylaminoethyl, 3-dimethylaminopropyl,piperidinyl and especially 4-piperidinyl, pyridinyl and especiallypyridine-3-yl or pyridin-4-yl, pyrrolidinyl and especiallypyrrolidin-3-yl, tetrahydrofuranyl and especially tetrahydro-furan-3-yl,tetrahydro-furan-2-ylmethyl, 4-chloro-benzyl, thiophen-2-yl-methyl, orR⁵ and R⁶ are both hydrogen, methyl, ethyl, or R⁵ and R⁶ together withthe N-atom to which they are bound form morpholine,4-aminocarbonyl-piperidine or azepane or —XSO₂NR⁵R⁶ is

R⁵ is more preferably selected from the group consisting of hydrogen,methyl, 2-hydroxyethyl, cyclobutyl, cyclopentyl, 2-dimethylaminoethyl,3-dimethylaminopropyl, tetrahydro-furan-3-yl, pyrrolidin-3-yl,pyridine-3-yl, pyridin-4-yl, and 4-piperidinyl.

R⁶ is more preferably hydrogen or methyl.

R⁸ is preferably C₁₋₄ alkyl, hydroxyl-C₂₋₄-alkyl; R⁸ is more preferablyhydroxy-C₂₋₄-alkyl, most preferably 2-hydroxyethyl.

R³ is preferably 1 to 3 substituents independently selected from thegroup consisting of hydrogen, halo, hydroxy, C₁₋₄alkyl, C₁₋₄ alkoxy,C₂₋₄ alkenyloxy, —OCF₃, C₂₋₄ alkanoyl, C₁₋₄alkylsulfonyl, mono- anddi-(C₁-C₄alkyl)sulfonamido, aminocarbonyl, mono- anddi-(C₁-C₄alkyl)aminocarbonyl, C₁₋₄ alkyloxymethyl,hydroxy-C₁₋₄alkyloxymethyl, cyano, —COOH and C₁-C₄ alkoxycarbonyl; orone substituent selected from C₃₋₇cycloalkyl, C₁₋₄ alkyl-cycloalkyl,C₁₋₄ alkyl-heterocycloalkyl, —O-heterocycloalkyl, aryl-C₁₋₄ alkoxy,heterocycloalkyl-C₁₋₄ alkoxy, heterocycloalkyl-C₁₋₄-alkyl,heteroaryl-C₁₋₄ alkoxy, heteroaryl-C₁₋₄-alkyl; wherein said substituentscan be further substituted by one or more radicals selected from thegroup of C₁₋₄-alkyl, hydroxyl-C₀₋₄-alkyl, C₁₋₄-alkoxy, halo,aminocarbonyl, and NR⁵R⁶. R³ is more preferably 1 to 3 substituentsindependently selected from the group consisting of methyl, ethyl,hydroxymethyl, hydroxy, methoxy, ethoxy, isopropoxy, benzyloxy,hydrogen, fluoro, chloro, trifluoromethyl, 2-methoxy-ethoxy,methoxymethyl, 2-methoxy-ethyl, tetrahydro-furan-3-yloxy,tetrahydro-furan-2-yl-methoxy, —N(CH₃)SO₂CH₃, piperidin-1-yl-methyl,2-hydroxymethyl-piperidin-1-yl-methyl,3-hydroxymethyl-piperidin-1-yl-methyl,3-(2-hydroxy-ethyl)-piperidin-1-yl-methyl,3-aminocarbonyl-piperidin-1-yl-methyl, dimethylaminomethyl,diethylaminomethyl, (ethyl-isopropyl-amino)-methyl,morpholin-4-ylmethyl, 4-methyl-piperazin-1-yl-methyl,[1,2,4]triazol-1-yl-methyl, pyridine-3-yl-methoxy andpyridine-4-yl-methoxy. R³ is most preferably methoxy, ethoxy, isopropoxyfluoro, diethylaminomethyl, 3-hydroxymethyl-piperidin-1-yl-methyl,morpholin-4-ylmethyl, piperidin-1-yl-methyl,4-methyl-piperazin-1-yl-methyl, and/or [1,2,4]triazol-1-yl-methyl.

Another preferred group of compounds of Formula I comprises those ofFormula Ia

wherein

R¹ is —CH₂SO₂NR⁵R⁶ or —CH₂SO₂R⁸;

R², R^(4a), R^(4b), R⁵, R⁶ and R⁸ are as defined above;R^(3a), R^(3b) and R^(3c) are as defined above for R³;and the N-oxide derivatives, prodrug derivatives, protected derivatives,individual isomers and mixtures of isomers thereof; and thepharmaceutically acceptable salts and solvates (e.g., hydrates) of suchcompounds.

Within these compounds of Formula Ia:

R² is preferably hydrogen.R^(3a) is preferably hydrogen or C₁₋₄ alkoxy; more preferably hydrogen,methoxy, ethoxy or isopropoxy, most preferably methoxy.R^(3b) is preferably hydrogen, C₁₋₄ alkyl optionally substituted bydialkylamine, heterocycloalkyl-C₁₋₄-alkyl optionally substituted byC₁₋₄-alkyl or by hydroxyl-C₀₋₄-alkyl, or heteroaryl-C₁₋₄-alkyl; R^(3b)is more preferably hydrogen, diethylaminomethyl,3-hydroxymethyl-piperidin-1-yl-methyl, morpholin-4-ylmethyl,piperidin-1-yl-methyl, 4-methyl-piperazin-1-yl-methyl, or[1,2,4]triazol-1-yl-methyl, most preferably hydrogen.R^(3c) is preferably hydrogen or halogen, especially fluoro; morepreferably hydrogen.R^(4a) and R^(4b) are preferably each independently C₁₋₄ alkyl orhydrogen; more preferably hydrogen or methyl; most preferably hydrogen.R⁵ is more preferably selected from the group consisting of hydrogen,methyl, 2-hydroxyethyl, cyclobutyl, cyclopentyl, 2-dimethylaminoethyl,3-dimethylaminopropyl, tetrahydro-furan-3-yl, pyrrolidin-3-yl,pyridine-3-yl, pyridin-4-yl and 4-piperidinyl, most preferably hydrogenor methyl.R⁶ is more preferably hydrogen or methyl.R⁸ is preferably hydroxy-C₂₋₄-alkyl, more preferably 2-hydroxyethyl.

The following compounds are preferred:

-   {4-[4-(2-Methoxy-phenyl)-pyrimidin-2-ylamino]-phenyl}-methanesulfonamide    (Compound 1);-   C-{4-[4-(2-Methoxy-phenyl)-pyrimidin-2-ylamino]-phenyl}-N-methyl-methanesulfonamide    (Compound 2); and-   {4-[4-(2-Methoxy-phenyl)-6-methyl-pyrimidin-2-ylamino]-phenyl}-methanesulfonamide    (Compound 3).

In a further embodiment, the invention provides the above mentionedcompounds according to Formula I, for medical use.

In a further embodiment, the invention provides pharmaceuticalcompositions containing a compound as outlined above, together with apharmaceutically acceptable carrier.

In a further embodiment, the invention provides use of a compound asoutlined above for preparing a pharmaceutical composition for treatinginflammatory disorders, immunological diseases, proliferative diseases,infectious diseases, cardiovascular diseases and neurodegenerativediseases.

In a further embodiment, the invention provides use of a compound asoutlined above for preparing a pharmaceutical composition for treatingpain, chronic pain, and/or neuropathic pain.

In a further embodiment, the invention provides a method for treatinginflammatory disorders, immunological diseases, proliferative diseases,infectious diseases, cardiovascular diseases and neurodegenerativediseases comprising the administration of an effective amount of atleast one of the compounds as mentioned above to a subject in needthereof.

In a further embodiment, the invention provides a method for treatingpain, chronic pain and/or neuropathic pain comprising the administrationof an effective amount of at least one of the compounds as mentionedabove to a subject in need thereof.

BRIEF DESCRIPTION OF THE DRAWING

FIG. 1 schematically depicts the spared nerve injury model (SNI model,as developed by Decosterd and Woolf (2000), which is characterized byligation and section of two branches of the sciatic nerve (namely tibialand common peroneal nerves) leaving the sural nerve intact.

DETAILED DESCRIPTION OF THE INVENTION

The inhibitors of CDK5 provided by the present invention are compoundsaccording to the general Formula I:

wherein

-   -   R¹ is —XSO₂NR⁵R⁶ or —XSO₂R⁸;    -   wherein X is a C₁₋₄ alkylene (including branched alkylene),        wherein said C₁₋₄ alkylene can be bound to R⁵ or R⁶ to form a 5-        or 6-membered heterocycle;    -   R⁵ and R⁶ independently of each other are hydrogen, C₁₋₄alkyl,        hydroxy-C₁₋₄alkyl or C₃₋₄alkenyl, C₃₋₈-cycloalkyl,        C₃₋₈-cycloalkyl-C₁₋₄alkyl or C₄₋₇heterocycloalkyl-C₀₋₄alkyl,        C₄₋₇aryl-C₀₋₄alkyl, C₄₋₇heteroaryl-C₀₋₄alkyl or    -   wherein R⁵ and R⁶ together with the N-atom to which they are        bound also may form a 5- to 8-membered heterocycloalkyl,    -   wherein said cycloalkyl, heterocycloalkyl, aryl, heteroaryl or        alkyl is further optionally substituted by up to 2 radicals        selected from the group consisting of halo, hydroxy,        aminocarbonyl, C₁₋₄ alkyl, hydroxy-C₁₋₄ alkyl, C₁₋₄ alkyl-O—C₁₋₄        alkyl, C₁₋₄ alkyl-O— and —NR⁵R⁶;    -   R⁸ is C₁₋₄ alkyl, hydroxy-C₂₋₄alkyl or C₃₋₄alkenyl,        C₃₋₈cycloalkyl, C₃₋₈-cycloalkyl-C₁₋₄alkyl or        C₄₋₇-heterocycloalkyl-C₀₋₄ alkyl;    -   wherein said cycloalkyl, heterocycloalkyl or alkyl is further        optionally substituted by up to 2 radicals selected from the        group consisting of halo, hydroxy, C₁₋₄ alkyl, hydroxy-C₁₋₄        alkyl, C₁₋₄ alkyl-O—C₁₋₄ alkyl, C₁₋₄ alkyl-O and —NR⁵R⁶;    -   R² is one or two substituents independently selected from        halogen and hydrogen;    -   R³ can be 1 to 3 substituents each independently selected from        the group consisting of hydrogen, halo, hydroxy, C₁₋₄alkyl,        C₃₋₇cycloalkyl, C₁₋₄ alkyl-cycloalkyl, C₁₋₄        alkyl-heterocycloalkyl, —O-heterocycloalkyl, C₁₋₄ alkoxy, C₂₋₄        alkenyloxy, —OCF₃, C₂₋₄ alkanoyl, C₁₋₄alkylsulfonyl, mono- and        di-(C₁-C₄alkyl)sulfonamido, aminocarbonyl, mono- and        di-(C₁-C₄alkyl)aminocarbonyl, aryl-C₁₋₄ alkoxy, heteroaryl-C₁₋₄        alkoxy, heterocycloalkyl-C1-4-alkoxy,        heterocycloalkyl-C1-4-alkyl, heteroaryl-C1-4-alkyl, C₁₋₄        alkyloxymethyl, hydroxy-C₁₋₄alkyloxymethyl, cyano, —COOH and        C₁-C₄ alkoxycarbonyl, wherein the above mentioned substituents        can be further substituted by radicals selected from the group        of C₁₋₄-alkyl, hydroxyl-C₀₋₄-alkyl, C₁₋₄-alkoxy, aminocarbonyl,        halo, and NR⁵R⁶;    -   R^(4a) and R^(4b) are the same or different and each is        independently hydrogen, C₁₋₄ alkyl or —NR′R″, wherein R′ and R″        are each independently hydrogen or C₁₋₄ alkyl;    -   and the N-oxide derivatives, prodrug derivatives, protected        derivatives, individual isomers and mixtures of isomers thereof;        and the pharmaceutically acceptable salts and solvates (e.g.,        hydrates) of such compounds.

The present invention thus provides new drugs or compounds, which drugsmay be used in therapeutic methods for the treatment of a CDK5-relateddisorder.

The present invention also provides compositions containing these newdrugs or compounds for modulating the activity of CDK5. The presentinvention further provides methods for performing rational drug designto develop drugs that can modulate activity of CDK5 and therebyameliorate a CDK5-related disorder.

The CDK5-inhibiting activity of a compound according to the presentinvention in a cell or tissue of interest can be determined by a methodcomprising: (a) determining a first level of cyclin-dependent kinase 5activity in said cell or tissue; (b) contacting said cell or tissue witha test compound; and (c) determining a second level of cyclin-dependentkinase 5 activity in said cell or tissue, wherein a difference in saidfirst level and said second level of cyclin-dependent kinase 5 activityis indicative of the ability of said test compound to modulatecyclin-dependent kinase 5 activity.

The present invention also provides diagnostic and therapeutic methodsfor the treatment of a CDK5-related disorder, including, but not limitedthe use of compositions or compounds of the invention in the treatmentof a CDK5-related disorder.

The invention provides methods of administering an agent (or drug orcompound) of the invention that can ameliorate a symptom of aCDK5-related disorder, disease and/or condition in a patient or subjectexhibiting the symptom. In certain embodiments, the invention providesmethods of administering an agent identified by the methods disclosedherein, that can ameliorate a symptom of a CDK5-related disorder in apatient or subject exhibiting the symptom.

As used herein, an “antagonist” is any compound that blocks thestimulation of a receptor and its resulting pharmacological effect.

As used herein, an “effective amount” of an inhibiting compound is anamount that can be determined by one of skill in the art based on datafrom studies using methods of analysis such as those disclosed herein.

As used herein, the terms “CDK5”, “Cdk5” or “cdk5” are usedinterchangeably with “cyclin-dependent kinase 5,” which is also known asneuronal cyclin-dependent-like protein (Nclk) and tau protein kinase II(TPKII). Cdk5 is a member of the cyclin dependent kinases but atypicallyCdk5 employs a non-cyclin cofactor called neuronal cyclin-dependent-likekinase 5 associated protein (Nck5a) rather than a cyclin.

As used herein, the term “CDK5-related disorder” is used interchangeablywith the terms “Cdk5 disorder,” “Cdk5 condition,” “Cdk5 dysfunction,”“Cdk5-related dysfunction,” “Cdk5-related disease,” “Cdk5-relatedcondition,” “dysregulation of Cdk5 function” or “Cdk5 functiondysregulation.” A Cdk5-related disorder includes, but is not limited to,depression, affective disorder, manic-depressive disorder,obsessive-compulsive disorder, eating disorder, emesis, panic disorder,anxiety disorder, migraine, myoclonus, premenstrual syndrome (PMS),post-traumatic stress syndrome, carcinoid syndrome, Alzheimer's disease,Huntington's Disease, Parkinson's disease, Tourette's syndrome, stroke,substance-induced psychotic disorder, for example psychosis induced byalcohol, amphetamine, cannabis, cocaine, hallucinogens, inhalants,opioids, or phencyclidine; personality disorder of the paranoid type;personality disorder of the schizoid type; drug addiction, includingnarcotic (e.g. heroin, opium, and morphine), cocaine and alcoholaddiction; drug withdrawal, including narcotic, cocaine and alcoholwithdrawal; epilepsy, sleep or circadian rhythm disorder (e.g.,insomnia), sexual disorder, stress disorder, schizophrenia, attentiondeficit disorder (ADD), attention deficit hyperactivity disorder (ADHD),neurodegenerative disorder, substance or drug abuse, pain (Pareek etal., PNAS 103, pp. 791-796; Pareek et al., Cell Cycle 5:6, pp. 585-588),and cancer. A Cdk5-related disorder also includes, but is not limitedto, a disease (e.g., depression) or a condition (e.g., addiction tococaine) that involves an aberration or dysregulation of a signaltransmission pathway, including but not limited to neurotransmissionmediated by serotonergic receptors in excitable cells, tissues or organs(e.g., neurons, brain, central nervous system, etc.). A Cdk5-relateddisorder also includes, but is not limited to, a symptom of aCdk5-related disorder. In certain embodiments, the pathway affectedincludes the phosphorylation and/or dephosphorylation of DARPP-32, withthe corresponding treatment of the dysregulation involving thestimulation and/or inhibition of the phosphorylation and/ordephosphorylation of one or more specific threonine and/or serineresidues of DARPP-32 (see, e.g., Greengard et al., Neuron 23:435-447(1999); Bibb et al., Proc. Natl Acad. Sci. USA 97:6809-68 14 (2000); andU.S. patent application Ser. Nos. 09/419,379, by Bibb et al., entitled“Methods of Identifying Agents That RegulatePhosphorylation/Dephosphorylation in Dopamine Signaling,” filed Oct. 15,1999, and Ser. No. 09/687,959, by Bibb et al., entitled “Methods ofIdentifying Agents That Regulate Phosphorylation/Dephosphorylation inDopamine Signaling,” filed Oct. 13, 2000, each of which is incorporatedherein by reference in its entirety).

Further diseases that are associated with abnormal cellular responsestriggered by protein kinase-mediated events, such as CDK5-mediatedevents, include autoimmune diseases, inflammatory diseases,cardiovascular diseases, allergies and hormone-related diseases. Inparticular, kinases have been implicated in various diseases including:diabetes and mood disorders such as bipolar disorder, cardiomyocetehypertrophy; and development and regulation of sperm motility. Further,kineses been implicated in hair loss and neurotrauma, for example,stroke, traumatic brain surgery and spinal cord trauma. These diseasesmay be caused by, or result in, the abnormal operation of cell signalingpathways in which CDK5 plays a role. Accordingly, molecules thatmodulate the activity of kinase-mediated signaling are useful astherapeutic agents in the treatment of such diseases.

Further examples of diseases or conditions where a kinase, such as CDK5may play a role include, cancer. The cancer may be a carcinoma, forexample carcinoma of the bladder, breast, colon, kidney, liver, lung,for example small cell lung cancer, esophagus, gall bladder, ovary,pancreas, stomach, cervix, thyroid, prostate, or skin, for examplesquamous cell carcinoma; a hematopoietic tumor of lymphoid lineage, forexample leukemia, acute lymphocytic leukemia, B-cell lymphoma, T-celllymphoma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, hairy celllymphoma, or Burkett's lymphoma; a hematopoietic tumor of myeloidlineage, for example acute and chronic myelogenous leukemias,myelodyplastic syndrome, or promyelocytic leukemia; a tumor ofmesenchymal origin, for example fibrosarcoma or rhabdomyosarcoma; atumor of the central or peripheral nervous system, for exampleastrocytoma, neuroblastomas, glioma or schwannoma; melanoma; seminoma;teratocarcinoma; osteosarcoma; xenoderoma pigmentoum; keratoctanthoma;thyroid follicular cancer; or Kaposi's sarcoma. Diseases or conditionscomprising benign abnormal cell growth include benign prostatehyperplasia, familial adenomatosis polyposis, neuro-fibromatosis,atherosclerosis, pulmonary fibrosis, arthritis, psoriasis,glomerulonephritis, restenosis, hypertrophic scar formation,inflammatory bowel disease, transplantation rejection, fungal infection,and endotoxic shock.

Pain

It has turned out that administration of CDK inhibitors according toFormula I have a hypoalgesic effect.

Thus, in a preferred embodiment, this invention relates to a method oftreating any type of pain comprising administering an effective amountof an inhibitor of CDK5 according to Formula I. Specifically, thecompounds of Formula I may be used for the treatment of chronic,neuropathic and/or inflammatory pain. In a particular preferredembodiment, the compounds of Formula I for use in the treatment of anytype of pain display an increased selectivity for CDK5 than for otherCDKs.

The role of CDK5 in the development of pain has been described in Pareeket al., PNAS 103, pp. 791-796 and in Pareek et al., Cell Cycle 5:6, pp.585-588.

The term “pain” as used herein generally relates to any type of pain andbroadly encompasses types of pain such as acute pain, chronic pain,inflammatory and neuropathic pain. In a preferred embodiment of thepresent invention, “pain” comprises neuropathic pain and associatedconditions. The pain may be chronic, allodynia (the perception of painfrom a normally innocuous stimulus), hyperalgesia (an exaggeratedresponse to any given pain stimulus) and an expansion of the receptivefield (i.e. the area that is “painful” when a stimulus is applied),phantom pain or inflammatory pain.

Acute pain types comprise, but are not limited to pain associated withtissue damage, postoperative pain, pain after trauma, pain caused byburns, pain caused by local or systemic infection, visceral painassociated with diseases comprising: pancreatitis, intestinal cystitis,dysmenorrhea, Irritable Bowel syndrome, Crohn's disease, ureteral colicand myocardial infarction.

Furthermore, the term “pain” comprises pain associated with CNSdisorders comprising: multiple sclerosis, spinal cord injury, traumaticbrain injury, Parkinson's disease and stroke.

In a preferred embodiment, “pain” relates to chronic pain typescomprising headache (for example migraine disorders, episodic andchronic tension-type headache, tension-type like headache, clusterheadache, and chronic paroxysmal hemicrania), low back pain, cancerpain, osteoarthritis pain and neuropathic pain, but is not limitedthereto.

Inflammatory pain (pain in response to tissue injury and the resultinginflammatory process) as defined herein relates to inflammatory painassociated with diseases comprising connective tissue diseases,rheumatoid arthritis, systemic lupus erythematosus, multiple sclerosisand arthritis, but is not limited thereto.

Neuropathic pain (pain resulting from damage to the peripheral nerves orto the central nervous system itself) includes conditions comprising,but not limited to metabolic neuropathies (e.g., diabetic neuropathy),post-herpetic neuralgia, trigeminal neuralgia, cranial neuralgia,post-stroke neuropathic pain, multiple sclerosis-associated neuropathicpain, HIV/AIDS-associated neuropathic pain, cancer-associatedneuropathic pain, carpal tunnel-associated neuropathic pain, spinal cordinjury-associated neuropathic pain, complex regional pain syndrome,fibromyalgia-associated neuropathic pain, reflex sympathic dystrophy,phantom limb syndrome or peripheral nerve or spinal cord trauma, nervetransection including surgery, limb amputation and stump pain, paincaused by the side effects of anti-cancer and anti-AIDS therapies,post-surgical neuropathic pain, neuropathy-associated pain such as inidiopathic or post-traumatic neuropathy and mononeuritis, andneuropathic pain caused by connective tissue disease such as rheumatoidarthritis, Wallenberg's syndrome, systemic lupus erythematosus, multiplesclerosis, or polyarteritis nodosa. The neuropathy can be classified asradiculopathy, mononeuropathy, mononeuropathy multiplex, polyneuropathyor plexopathy.

The term “allodynia” denotes pain arising from stimuli which are notnormally painful. Allodynic pain may occur other than in the areastimulated.

The term “hyperalgesia” denotes an increased sensitivity to a painfulstimulus.

The term “hypoalgesia” denotes a decreased sensitivity to a painfulstimulus.

Inflammatory Diseases

Surprisingly, it could be shown that the CDK inhibitors according toFormula I as disclosed herein exert an anti-inflammatory effect in invitro and in vivo inflammatory assays.

Thus, in a preferred embodiment, this invention relates to a method oftreating inflammatory diseases comprising administering an effectiveamount of an inhibitor of cyclin-dependent kinase according to FormulaI. In a particular preferred embodiment, the compounds of Formula I foruse in the treatment of inflammatory diseases display an increasedselectivity for CDK5 than for other CDKs.

Thus, the compounds according to Formula I as presented herein may beused for the treatment and/or prevention of inflammatory diseases.

The term “inflammatory diseases” as used herein relates to diseasestriggered by cellular or non-cellular mediators of the immune system ortissues causing the inflammation of body tissues and subsequentlyproducing an acute or chronic inflammatory condition.

Examples of inflammatory diseases are hypersensitivity reactions of typeI-IV, for example but not limited to hypersensitivity diseases of thelung including asthma, atopic diseases, allergic rhinitis orconjunctivitis, angioedema of the lids, hereditary angioedema,antireceptor hypersensitivity reactions and autoimmune diseases,Hashimoto's thyroiditis, systemic lupus erythematosus, Goodpasture'ssyndrome, pemphigus, myasthenia gravis, Grave's and Raynaud's disease,type B insulin-resistant diabetes, rheumatoid arthritis, psoriasis,Crohn's disease, scleroderma, mixed connective tissue disease,polymyositis, sarcoidosis, Wegener's granulomatosis, glomerulonephritis,acute or chronic host versus graft reactions.

Furthermore, the term “inflammatory diseases” includes but is notlimited to abdominal cavity inflammation, dermatitis, gastrointestinalinflammation (including inflammatory bowel disease, ulcerative colitis),fibrosis, ocular and orbital inflammation, dry eye disease and severedry eye disease resulting from Sjörgen's syndrome, mastitis, otitis,mouth inflammation, musculoskeletal system inflammation (including gout,osteoarthritis), inflammatory diseases of the central nervous system(including multiple sclerosis, bacterial meningitis, meningitis),genitourinary tract inflammation (incl prostatitis, glomerulonephritis),cardiovascular inflammation (including atherosclerosis, heart failure),respiratory tract inflammation (including chronic bronchitis, chronicobstructive pulmonary disease), thyroiditis, diabetes mellitus,osteitis, myositis, multiple organ failure (including, sepsis),polymyositis and psoriatic arthritis.

immunological diseases

The compounds according to Formula I are also envisaged to be useful inthe treatment and/or prevention of immunological diseases, such as, forexample, autoimmune diseases.

Accordingly, the present invention provides a method for the treatmentand/or prevention of immunological diseases comprising theadministration of an effective amount of at least one CDK5 inhibitoraccording to Formula I to a subject in need thereof.

The term “immunological diseases” as used herein relates to diseasesincluding but not limited to allergy, asthma, graft-versus-host disease,immune deficiencies and autoimmune diseases.

Specifically, immunological diseases include diabetes, rheumaticdiseases, AIDS, chronic granulomatosis disease, rejection oftransplanted organs and tissues, rhinitis, chronic obstructive pulmonarydiseases, osteoporosis, ulcerative colitis, Crohn's disease, sinusitis,lupus erythematosus, psoriasis, multiple sclerosis, myasthenia gravis,alopecia, recurrent infections, atopic dermatitis, eczema and severeanaphylactic reactions, but are not limited thereto. Furthermore,“immunological diseases” also include allergies such as contactallergies, food allergies or drug allergies.

Proliferative Diseases

The compounds of Formula I are inhibitors of cyclin-dependent kinases,which represent key molecules involved in regulation of the cell cycle.Cell-cycle disregulation is one of the cardinal characteristics ofneoplastic cells. Thus, said compounds are expected to prove useful inarresting or recovering control of the cell cycle in abnormally dividingcells. It is thus expected that the compounds according to Formula I areuseful in the treatment and/or prevention of proliferative diseases suchas cancer.

Accordingly, the invention provides a method for the treatment and/orprevention of proliferative diseases comprising administering aneffective amount of at least one inhibitor of a cyclin-dependent kinaseaccording to Formula I.

As used herein, the term “proliferative disease” relates to cancerdisorders, including, but not limited to benign neoplasms, dysplasias,hyperplasias as well as neoplasms showing metastatic growth or any othertransformations.

The term “cancer” includes but is not limited to benign and malignneoplasia like carcinoma, sarcoma, carcinosarcoma, cancers of theblood-forming tissues, tumors of nerve tissues including the brain andcancer of skin cells.

Examples of cancers which may be treated include, but are not limitedto, a carcinoma, for example a carcinoma of the bladder, breast, colon(e.g. colorectal carcinomas such as colon adenocarcinoma and colonadenoma), kidney, epidermal, liver, lung, for example adenocarcinoma,small cell lung cancer and non-small cell lung carcinomas, oesophagus,gall bladder, ovary, pancreas e.g. exocrine pancreatic carcinoma,stomach, cervix, thyroid, prostate, or skin, for example squamous cellcarcinoma; a hematopoietic tumour of lymphoid lineage, for exampleleukemia, acute lymphocytic leukemia, B-cell lymphoma, T-cell lymphoma,Hodgkin's lymphoma, non-Hodgkin's lymphoma, hairy cell lymphoma, orBurkett's lymphoma; a hematopoietic tumor of myeloid lineage, forexample acute and chronicmyelogenous leukemias, myelodysplasticsyndrome, or promyelocytic leukemia; thyroid follicular cancer; a tumourof mesenchymal origin, for example fibrosarcoma or habdomyosarcoma; atumor of the central or peripheral nervous system, for exampleastrocytoma, neuroblastoma, glioma or schwannoma; melanoma; seminoma;teratocarcinoma; osteosarcoma; xenoderoma pigmentoum; keratoctanthoma;thyroid follicular cancer; Kaposi's sarcoma, astrocytoma, basal cellcarcinoma, small intestine cancer, small intestinal tumors,gastrointestinal tumors, glioblastomas, liposarcoma, germ cell tumor,head and neck tumors (tumors of the ear, nose and throat area), cancerof the mouth, throat, larynx, and the esophagus, cancer of the bone andits supportive and connective tissues like malignant or benign bonetumour, e.g. malignant osteogenic sarcoma, benign osteoma, cartilagetumors; like malignant chondrosarcoma or benign chondroma,osteosarcomas; tumors of the urinary bladder and the internal andexternal organs and structures of the urogenital system of male andfemale, soft tissue tumors, soft tissue sarcoma, Wilm's tumor or cancersof the endocrine and exocrine glands like e.g. thyroid, parathyroid,pituitary, adrenal glands, salivary glands.

Cardiovascular Diseases

Furthermore, the invention relates to the treatment and/or prevention ofcardiovascular diseases comprising administering an effective amount ofat least one inhibitor of a cyclin-dependent kinase according to FormulaI.

It has been reported that the field of cariodvascular diseasesconstitutes a possible clinical application for CDK inhibitors(Pharmacol Ther 1999, 82(2-3):279-284).

Thus, in a preferred embodiment, the invention relates to a method oftreating and/or preventing cardiovascular diseases comprisingadministering an effective amount of at least one inhibitor of acyclin-dependent kinase according to Formula I, wherein said compounddisplays an increased selectivity for CDK5 than for other CDKs.

The term “cardiovascular diseases” includes but is not limited todisorders of the heart and the vascular system like congestive heartfailure, myocardial infarction, ischemic diseases of the heart, such asstable angina, unstable angina and asymptomatic ischemia, all kinds ofatrial and ventricular arrhythmias, hypertensive vascular diseases,peripheral vascular diseases, coronary heart disease andatherosclerosis. Furthermore, as used herein, the term includes, but isnot limited to adult congenital heart disease, aneurysm, anginapectoris, angioneurotic edema, aortic valve stenosis, aortic aneurysm,aortic regurgitation, arrhythmogenic right ventricular dysplasia,arteriovenous malformations, atrial fibrillation, Behcet syndrome,bradycardia, cardiomegaly, cardiomyopathies such as congestive,hypertrophic and restrictive cardiomyopathy, carotid stenosis, cerebralhemorrhage, Churg-Strauss syndrome, cholesterol embolism, bacterialendocarditis, fibromuscular dysplasia, congestive heart failure, heartvalve diseases such as incompetent valves or stenosed valves, heartattack, epidural or subdural hematoma, von Hippel-Lindau disease,hyperemia, hypertension, pulmonary hypertension, hypertrophic growth,left ventricular hypertrophy, right ventricular hypertrophy, hypoplasticleft heart syndrome, hypotension, intermittent claudication, ischemicheart disease, Klippel-Trenaunay-Weber syndrome, lateral medullarysyndrome, mitral valve prolapse, long QT syndrome mitral valve prolapse,myocardial ischemia, myocarditis, disorders of the pericardium,pericarditis, peripheral vascular diseases, phlebitis, polyarteritisnodosa, pulmonary atresia, Raynaud disease, restenosis, rheumatic heartdisease, Sneddon syndrome, stenosis, superior vena cava syndrome,syndrome X, tachycardia, hereditary hemorrhagic telangiectasia,telangiectasis, temporal arteritis, thromboangiitis obliterans,thrombosis, thromboembolism, varicose veins, vascular diseases,vasculitis, vasospasm, ventricular fibrillation, Williams syndrome,peripheral vascular disease, varicose veins and leg ulcers, deep veinthrombosis and Wolff-Parkinson-White syndrome.

Furthermore, the term cardiovascular diseases includes diseasesresulting from congenital defects, genetic defects, environmentalinfluences (i.e., dietary influences, lifestyle, stress, etc.), andother defects or influences.

Neurodegenerative Diseases

CDK inhibitors have been described to exert neuroprotective effects.Specifically, it has been reported that CDK inhibitors prevent neuronaldeath in neurodegenerative diseases such as Alzheimer's disease (BiochemBiophys Res Commun 2002 (297):1154-1158; Trends Pharmacol Sci 2002(23):417-425; Pharmacol Ther 1999, 82(2-3):279-284).

Thus, the compounds according to Formula I, which are CDK5 inhibitors,are expected to provide beneficial effects in the therapeutic managementof neurodegenerative diseases.

Accordingly, the invention relates a method of treating and/orpreventing neurodegenerative diseases comprising administering aneffective amount of at least one inhibitor of a cyclin-dependent kinaseaccording to Formula I.

The term “neurodegenerative diseases” as used herein includes disordersof the central nervous system as well as disorders of the peripheralnervous system, including, but not limited to brain injuries,cerebrovascular diseases and their consequences, Parkinson's disease,corticobasal degeneration, motor neuron disease, dementia, includingALS, multiple sclerosis, traumatic brain Injury, stroke, post-stroke,post-traumatic brain injury, and small-vessel cerebrovascular disease,dementias, such as Alzheimer's disease, vascular dementia, dementia withLewy bodies, frontotemporal dementia and Parkinsonism linked tochromosome 17, frontotemporal dementias, including Pick's disease,progressive nuclear palsy, corticobasal degeneration, Huntington'sdisease, thalamic degeneration, Creutzfeld-Jakob dementia, HIV dementia,schizophrenia with dementia, Korsakoffs psychosis and AIDS-relateddementia.

Similarly, cognitive-related disorders, such as mild cognitiveimpairment, age-associated memory impairment, age-related cognitivedecline, vascular cognitive impairment, attention deficit disorders,attention deficit hyperactivity disorders, and memory disturbances inchildren with learning disabilities are also considered to beneurodegenerative disorders.

Specifically, the present invention relates to a method for treating theabove-referenced types of pain and associated conditions andinflammatory disorders, immunological diseases, proliferative diseases,infectious diseases, cardiovascular diseases and neurodegenerativediseases, wherein the term “treating” comprises the prevention,amelioration or treating, of pain and associated conditions andinflammatory disorders, immunological diseases, proliferative diseases,infectious diseases, cardiovascular diseases and neurodegenerativediseases

Pharmaceutical Compositions

Preferred embodiments of the present invention include theadministration of compositions comprising at least one cyclin-dependentkinase inhibitor according to Formula I as an active ingredient togetherwith at least one pharmaceutically acceptable (i.e. non-toxic) carrier,excipient and/or diluent.

Preferably, the composition comprises at least one cyclin-dependentkinase inhibitor according to Formula I as an active ingredient, whereinsaid at least one cyclin-dependent kinase inhibitor has an increasedselectivity for CDK5 than for other CDKs.

Furthermore, the invention also comprises compositions combining atleast two inhibitors of CDK and/or pharmaceutically acceptable saltsthereof. Said at least two inhibitors may inhibit the samecyclin-dependent kinase or may also inhibit different types ofcyclin-dependent kinases, e.g. one inhibitor in the composition mayinhibit CDK5 while the other inhibitor is capable of inhibiting CDK2,for example. However, one of the inhibitors is always a CDK5 inhibitor.

Having regard to pain treatment, an individual pain medication oftenprovides only partially effective pain alleviation because it interfereswith just one pain-transducing pathway out of many. Thus, it is alsointended to administer CDK inhibitors according to Formula I incombination with a pain-reducing (analgesic) agent that acts at adifferent point in the pain perception process.

An “analgesic agent” comprises a molecule or combination of moleculesthat causes a reduction in pain perception. An analgesic agent employs amechanism of action other than inhibition of CDK.

One class of analgesics, such as nonsteroidal anti-inflammatory drugs(NSAIDs), down-regulates the chemical messengers of the stimuli that aredetected by the nociceptors and another class of drugs, such as opioids,alters the processing of nociceptive information in the CNS. Otheranalgesics are local anesthetics, anticonvulsants and antidepressantssuch as tricyclic antidepressants. Administering one or more classes ofdrug in addition to CDK inhibitors can provide more effectiveamelioration of pain.

Preferred NSAIDs for use in the methods and compositions of the presentinvention are aspirin, acetaminophen, ibuprofen, and indomethacin.Furthermore, cyclooxygenase-2 (COX-2) inhibitors, such as specific COX-2inhibitors (e.g. celecoxib, COX189, and rofecoxib) may also be used asan analgesic agent in the methods or compositions of the presentinvention.

Preferred tricyclic antidepressants are selected from the groupconsisting of Clomipramine, Amoxapine, Nortriptyline, Amitriptyline,Imipramine, Desipramine, Doxepin, Trimipramine, Protriptylin, andImipramine pamoate.

Furthermore, the use of anticonvulsants (e.g. gabapentin), GABABagonists (e.g. L-baclofen), opioids, vanniloid receptor antagonists andcannabinoid (CB) receptor agonists, e.g. CB1 receptor agonists asanalgesic is also preferred in the methods and compositions in thepresent invention.

In preparing cyclin-dependent kinase inhibitor compositions of thisinvention, one can follow the standard recommendations of well-knownpharmaceutical sources such as Remington: The Science and Practice ofPharmacy, ^(19th) ed. (Mack Publishing, 1995).

The pharmaceutical compositions of the present invention can be preparedin a conventional solid or liquid carrier or diluent and a conventionalpharmaceutically-made adjuvant at suitable dosage level in a known way.The preferred preparations are adapted for oral application. Theseadministration forms include, for example, pills, tablets, film tablets,coated tablets, capsules, powders and deposits.

Furthermore, the present invention also includes pharmaceuticalpreparations for parenteral application, including dermal, intradermal,intragastral, intracutan, intravasal, intravenous, intramuscular,intraperitoneal, intranasal, intravaginal, intrabuccal, percutan,rectal, subcutaneous, sublingual, topical, or transdermal application,wherein said preparations in addition to typical vehicles and/ordiluents contain at least one inhibitor according to the presentinvention and/or a pharmaceutical acceptable salt thereof as activeingredient.

The pharmaceutical compositions according to the present inventioncontaining at least one inhibitor according to the present inventionand/or a pharmaceutical acceptable salt thereof as active ingredientwill typically be administered together with suitable carrier materialsselected with respect to the intended form of administration, i.e. fororal administration in the form of tablets, capsules (either solidfilled, semi-solid filled or liquid filled), powders for constitution,gels, elixirs, dispersable granules, syrups, suspensions, and the like,and consistent with conventional pharmaceutical practices. For example,for oral administration in the form of tablets or capsules, the activedrug component may be combined with any oral non-toxic pharmaceuticallyacceptable carrier, preferably with an inert carrier like lactose,starch, sucrose, cellulose, magnesium stearate, dicalcium phosphate,calcium sulfate, talc, mannitol, ethyl alcohol (liquid filled capsules)and the like.

Moreover, suitable binders, lubricants, disintegrating agents andcoloring agents may also be incorporated into the tablet or capsule.Powders and tablets may contain about 5 to about 95% by weight of acyclin-dependent kinase inhibitor according to the Formula I as recitedherein or analogues thereof or the respective pharmaceutical active saltas active ingredient.

Suitable binders include starch, gelatin, natural sugars, cornsweeteners, natural and synthetic gums such as acacia, sodium alginate,carboxymethylcellulose, polyethylene glycol and waxes. Among suitablelubricants there may be mentioned boric acid, sodium benzoate, sodiumacetate, sodium chloride, and the like.

Suitable disintegrants include starch, methylcellulose, guar gum, andthe like.

Sweetening and flavoring agents as well as preservatives may also beincluded, where appropriate. The disintegrants, diluents, lubricants,binders etc. are discussed in more detail below.

Moreover, the pharmaceutical compositions of the present invention maybe formulated in sustained release form to provide the rate controlledrelease of any one or more of the components or active ingredients tooptimise the therapeutic effect (s), e.g. antihistaminic activity andthe like. Suitable dosage forms for sustained release include tabletshaving layers of varying disintegration rates or controlled releasepolymeric matrices impregnated with the active components and shaped intablet form or capsules containing such impregnated or encapsulatedporous polymeric matrices.

Liquid form preparations include solutions, suspensions, and emulsions.As an example, there may be mentioned water or water/propylene glycolsolutions for parenteral injections or addition of sweeteners andopacifiers for oral solutions, suspensions, and emulsions. Liquid formpreparations may also include solutions for intranasal administration.

Aerosol preparations suitable for inhalation may include solutions andsolids in powder form, which may be present in combination with apharmaceutically acceptable carrier such as an inert, compressed gas,e.g. nitrogen.

For preparing suppositories, a low melting wax, such as a mixture offatty acid glycerides like cocoa butter is melted first, and the activeingredient is then dispersed homogeneously therein e.g. by stirring. Themolten, homogeneous mixture is then poured into conveniently sizedmoulds, allowed to cool, and thereby solidified.

Also included are solid form preparations, which are intended to beconverted, shortly before use, to liquid form preparations for eitheroral or parenteral administration. Such liquid forms include solutions,suspensions, and emulsions.

The compounds according to the present invention may also be deliveredtransdermally. The transdermal compositions may have the form of acream, a lotion, an aerosol and/or an emulsion and may be included in atransdermal patch of the matrix or reservoir type as is known in the artfor this purpose.

The term capsule as recited herein refers to a specific container orenclosure made e.g. of methylcellulose, polyvinyl alcohols, or denaturedgelatins or starch for holding or containing compositions comprising theactive ingredient(s). Capsules with hard shells are typically made ofblended or relatively high gel strength gelatins from bones or porkskin. The capsule itself may contain small amounts of dyes, opaquingagents, plasticisers and/or preservatives. Under tablet a compressed ormoulded solid dosage form is understood which comprises the activeingredients with suitable diluents. The tablet may be prepared bycompression of mixtures or granulations obtained by wet granulation, drygranulation, or by compaction well known to a person of ordinary skillin the art.

Oral gels refer to the active ingredients dispersed or solubilised in ahydrophilic semi-solid matrix.

Powders for constitution refers to powder blends containing the activeingredients and suitable diluents which can be suspended e.g. in wateror in juice.

Suitable diluents are substances that usually make up the major portionof the composition or dosage form. Suitable diluents include sugars suchas lactose, sucrose, mannitol, and sorbitol, starches derived fromwheat, corn rice, and potato, and celluloses such as microcrystallinecellulose. The amount of diluent in the composition can range from about5 to about 95% by weight of the total composition, preferably from about25 to about 75% by weight, and more preferably from about 30 to about60% by weight.

The term disintegrants refers to materials added to the composition tosupport disintegration and release of the pharmaceutically activeingredients of a medicament. Suitable disintegrants include starches,“cold water soluble” modified starches such as sodium carboxymethylstarch, natural and synthetic gums such as locust bean, karaya, guar,tragacanth and agar, cellulose derivatives such as methylcellulose andsodium carboxymethylcellulose, microcrystalline celluloses, andcross-linked microcrystalline celluloses such as sodiumcroscaramellose,alginates such as alginic acid and sodium alginate, clays such asbentonites, and effervescent mixtures. The amount of disintegrant in thecomposition may range from about 2 to about 20% by weight of thecomposition, more preferably from about 5 to about 10% by weight.

Binders are substances which bind or “glue” together powder particlesand make them cohesive by forming granules, thus serving as the“adhesive” in the formulation. Binders add cohesive strength alreadyavailable in the diluent or bulking agent. Suitable binders includesugars such as sucrose, starches derived from wheat corn rice andpotato, natural gums such as acacia, gelatin and tragacanth, derivativesof seaweed such as alginic acid, sodium alginate and ammonium calciumalginate, cellulose materials such as methylcellulose, sodiumcarboxymethylcellulose and hydroxypropylmethylcellulose,polyvinylpyrrolidone, and inorganic compounds such as magnesium aluminumsilicate. The amount of binder in the composition may range from about 2to about 20% by weight of the composition, preferably from about 3 toabout 10% by weight, and more preferably from about 3 to about 6% byweight.

Lubricants refer to a class of substances which are added to the dosageform to enable the tablet granules etc. after being compressed torelease from the mould or die by reducing friction or wear. Suitablelubricants include metallic stearates such as magnesium stearate,calcium stearate, or potassium stearate, stearic acid, high meltingpoint waxes, and other water soluble lubricants such as sodium chloride,sodium benzoate, sodium acetate, sodium oleate, polyethylene glycols andD,L-leucine. Lubricants are usually added at the very last step beforecompression, since they must be present at the surface of the granules.The amount of lubricant in the composition may range from about 0.2 toabout 5% by weight of the composition, preferably from about 0.5 toabout 2% by weight, and more preferably from about 0.3 to about 1.5% byweight of the composition.

Glidents are materials that prevent baking of the components of thepharmaceutical composition together and improve the flow characteristicsof granulate so that flow is smooth and uniform. Suitable glidentsinclude silicon dioxide and talc.

The amount of glident in the composition may range from about 0.1 toabout 5% by weight of the final composition, preferably from about 0.5to about 2% by weight.

Coloring agents are excipients that provide coloration to thecomposition or the dosage form. Such excipients can include food gradedyes adsorbed onto a suitable adsorbent such as clay or aluminum oxide.The amount of the coloring agent may vary from about 0.1 to about 5% byweight of the composition, preferably from about 0.1 to about 1% byweight.

The present invention relates to the administration of compositionscontaining as active ingredient a cyclin-dependent kinase inhibitor to asubject in need thereof for the treatment of any type of pain,inflammatory disorders, immunological diseases, proliferative diseases,cardiovascular diseases or neurodegenerative diseases.

“A subject in need thereof” comprises an animal, preferably a mammal,and most preferably a human, expected to experience any type of pain,inflammatory disorders, immunological diseases, proliferative diseases,cardiovascular diseases or neurodegenerative diseases in the near futureor which has ongoing experience of said conditions. For example, suchanimal or human may have a ongoing condition that is causing paincurrently and is likely to continue to cause pain, or the animal orhuman has been, is or will be enduring a procedure or event that usuallyhas painful consequences. Chronic painful conditions such as diabeticneuropathic hyperalgesia and collagen vascular diseases are examples ofthe first type; dental work, particularly in an area of inflammation ornerve damage, and toxin exposure (including exposure to chemotherapeuticagents) are examples of the latter type.

In order to achieve the desired therapeutic effect, the respectivecyclin-dependent kinase inhibitor has to be administered in atherapeutically effective amount.

The term “therapeutically effective amount” is used to indicate anamount of an active compound, or pharmaceutical agent, that elicits thebiological or medicinal response indicated. This response may occur in atissue, system, animal or human that is being sought by a researcher,veterinarian, medical doctor or other clinician, and includesalleviation of the symptoms of the disease being treated. In the contextof the present invention, a therapeutically effective amount comprises,e.g., an amount that reduces pain, in particular inflammatory orneuropathic pain. Specifically, a therapeutically effective amountdenotes an amount which exerts a hypoalgesic effect in the subject to betreated.

Such effective amount will vary from subject to subject depending on thesubject's normal sensitivity to, e.g., pain, its height, weight, age,and health, the source of the pain, the mode of administering theinhibitor of CDKs, the particular inhibitor administered, and otherfactors. As a result, it is advisable to empirically determine aneffective amount for a particular subject under a particular set ofcircumstances.

The invention is further illustrated by the following non-limitingexamples.

EXAMPLES

All reagents were purchased from ACROS Organics, Aldrich, Lancaster,Maybridge and Boron Molecular.

The LC/MS analyses for the compounds were done at Surveyor MSQ (ThermoFinnigan, USA) with APCI ionization.

The compounds of the present invention can be prepared by any methodknown in the art. One convenient synthetic route is shown below inScheme 1:

Palladium-catalyzed cross-coupling of phenyl boronic acids (2, Y═B(OH)₂)or their derivatives with 2,4-dihalogenated pyrimidines (1, e.g.X¹═X²═Cl) affords 4-arylated 2-halogenopyrimidines (3), which areaminated with anilines (4).

Example 1 Synthesis of{4-[4-(2-Methoxy-phenyl)-pyrimidin-2-ylamino]-phenyl}-methanesulfonamide(Compound 1)

a. 4,6-Dichloro-pyrimidine (6 g, 0.04 mol) (Ranganathan, Subramania etal., Proceedings—Indian Academy of Sciences, Chemical Sciences (1994),106(5), 1051-70. Maggiali, C. et al., Farmaco, Edizione Scientifica(1988), 43(3), 277-91. Gershon, Herman et al., Journal of Med. Chem.(1963), 6, 87-9) and (2-methoxy-phenyl)-boronic acid (4.37 g, 0.029 mol)were dissolved in dimethoxyethane (120 mL) and water (18 mL). To thissolution were added NaHCO₃ (6.72 g, 0.08 mol), PdCl₂(PPh₃)₂ (0.84 g) andallowed to reflux for 7 hrs (TLC control). Then the mixture was cooleddown to room temperature and the solvents removed under reducedpressure. The obtained crude solid was dissolved in dichloromethane (100mL), washed with water (1×100 mL), the organic layer was separated,dried over K₂CO₃, filtered and the solvent removed under reducedpressure. The obtained solid was further purified byflash-chromatography (eluent dichloromethane) to afford a crude productthat was crystallized from hexane to yield2-chloro-4-(2-methoxy-phenyl)-pyrimidine (4.7 g, 73%).b. A solution of 2-chloro-4-(2-methoxy-phenyl)-pyrimidine (0.883 g,0.004 mol), and (4-amino-phenyl)-methanesulfonamide (0.745 g, 0.004 mol)(Bosch, J.; Roca, T. et al., Tetrahedron (2001), 57(6), 1041-1048. Owa,Takashi et al., Journal of Med. Chem. (1999), 42(19), 3789-3799) in DMF(12 ml) was stirred for 2 hours at 80° C. (TLC control). The solvent wasremoved under reduced pressure to afford{4-[4-(2-Methoxy-phenyl)-pyrimidin-2-ylamino]-phenyl}-methanesulfonamide.

Example 2 Synthesis ofC-{4-[4-(2-Methoxy-phenyl)-pyrimidin-2-ylamino]-phenyl}-N-methyl-methanesulfonamide(Compound 2)

A solution of 2-chloro-4-(2-methoxy-phenyl)-pyrimidine (0.883 g, 0.004mol), and C-(4-amino-phenyl)-N-methyl-methanesulfonamide (0.801 g, 0.004mol) (Macor, John E.; Blank et al., Tetrahedron Letters (1992), 33(52),8011-14) in DMF is stirred in DMF (12 ml) for 2 hours at 80° C. (TLCcontrol). The solvent was removed under reduced pressure to giveC-{4-[4-(2-methoxy-phenyl)-pyrimidin-2-ylamino]-phenyl}-N-methyl-methanesulfonamide.

Example 3 Synthesis of{4-[4-(2-Methoxy-phenyl)-6-methyl-pyrimidin-2-ylamino]-phenyl}-methanesulfonamide(Compound 3)

A solution of 2-chloro-4-(2-methoxy-phenyl)-6-methyl-pyrimidine (0.939g, 0.004 mol) (Cocuzza, Anthony J. et al., PCT Int. Appl. (1999), WO9901439 A1, 19990114.), and (4-amino-phenyl)-methanesulfonamide (0.745g, 0.004 mol) in DMF (12 ml) is stirred for 2 hours at 80° C. (TLCcontrol). The solvent was removed under reduced pressure to give{4-[4-(2-methoxy-phenyl)-6-methyl-pyrimidin-2-ylamino]-phenyl}-methanesulfonamide.

Compound No. Structure IUPAC name MS m/z 1

{4-[4-(2-Methoxy-phenyl)-pyrimidin-2-ylamino]-phenyl}-methanesulfonamide 371.9 (M + 1) 2

C-{4-[4-(2-Methoxy-phenyl)-pyrimidin- 2-ylamino]-phenyl}-N-methyl-methanesulfonamide 385, 378 (M + 1) 3

{4-[4-(2-Methoxy-phenyl)-6-methyl- pyrimidin-2-ylamino]-phenyl}-methanesulfonamide 385.9 (M + 1)

Example 4 I. Behavioral Animal Models for the Analysis of Inflammatoryand Neuropathic Pain

Several animal models for the analysis of inflammatory and neuropathicpain are known. Said models share the common feature that after e.g.,induction of a nerve lesion (e.g., spared nerve injury, SNI) or afterexposing experimental animals to a noxious stimulus (e.g., injection offormalin or carrageenan), the signs of pain as induced by saidinterventions are measured by quantifiable behavioral components suchas, e.g., paw withdrawal threshold to mechanical stimulation with vonFrey hairs (or to thermal stimulation using a laser source or lickingbehaviour). These reactions are interpreted as being equivalent tomechanical and thermal allodynia (hypersensitivity to mechanicalstimuli) or hyperalgesia in humans.

The spared nerve injury model (SNI model, as developed by Decosterd andWoolf (2000), see FIG. 1) is characterized by the induction ofclinically relevant nerve lesions and after surgical intervention,subsequent behavioral experiments (e.g., von Frey Assay). Said modelconstitutes a common nerve injury model which consists of ligation andsection of two branches of the sciatic nerve (namely tibial and commonperoneal nerves) leaving the sural nerve intact. The SNI model resultsin early (less than 24 hours), prolonged and substantial changes inmechanical and cold sensitivity that closely mimic the features ofclinical neuropathic pain. Animals with these types of nerve injury havebeen shown to develop abnormal pain sensations and hypersensitivity tomechanical stimuli (allodynia) similar to those reported by neuropathicpain patients.

Alternatively, the formalin assay in mice is a valid and reliablebehavioral model of nociception in inflammatory and neuropathic pain. Itis sensitive to various classes of analgesic drugs (Hunskaar S, Hole K,Pain. 1987 July; 30(1):103-14.) The noxious stimulus consists of aninjection of 10 μl diluted formalin (2% in saline) under the skin of thedorsal surface of the left hindpaw (subcutaneous or interplantar intothe left hindpaw). The response is licking and flinching of the injectedpaw.

For the carrageenan assay a subcutaneous injection of 25 μl of 1%carrageenan (in saline) into a single hind paw (ipsi-lateral paw) ofmice is applied. Subsequent inflammation results in long lastingswelling and hypersensitivity (against mechanical and thermal stimuli)of the paw. The carrageenan assay is a standard laboratory assay used topredict anti-inflammatory activity of test compounds. Paw edemameasurements and Hargreaves Assay (withdrawal of paws due to thermalstimulation via a light source) are used for read out.

Regarding the present invention, the effect of administration ofcyclin-dependent kinase (CDK)-inhibiting compounds according to FormulaI on the development of inflammatory and neuropathic pain is assayed ina SNI model, in a carrageenan and in a formalin assay. The experimentalprocedure and results are described in detail below.

Example 5

A. Spared Nerve Injury (SNI)—Model of Chronic Neuropathic Pain

As outlined above, the spared nerve injury (SNI) model (see FIG. 1)involves a lesion of two of the three terminal branches of the sciaticnerve (tibial and common peroneal nerves) of experimental animals,leaving the sural nerve intact. SNI results in mechanical and thermalallodynia in the non-injured sural nerve skin territory (Decosterd andWoolf, Pain 2000; 87:149-158. (2) Tsujino et al., Mol. Cel. Neurosci.2000; 15:170-182).

1. Induction of Spared Nerve Injury (Nerve Lesion) in Wildtype Mice

Wildtype mice (strain C3HeB/FeJ) (age, sex and weight matched) wereanesthetized with Hypnorm (0.315 mg/ml fentanyl citrate+10 mg/mlfluanisone; Janssen)/Hypnovel (5 mg/ml midazolam; Roche AppliedSciences)/water at a ratio of 1:1:2 at 4 μl/g prior to surgicalpreparation.

Subsequently, an incision was made under aseptic precautions in theipsi-lateral right hind leg of all mice just above the level of theknee, exposing the three terminal branches of the sciatic nerve: thecommon peroneal, tibial, and sural nerves. The common peroneal andtibial nerves were ligated tightly with 7/0 silk and sectioned distal tothe ligation removing ≈2 mm of distal nerve stump. The sural branchremained untouched during the procedure (denoted herein “SNI ipsi”). Theoverlying muscle and skin was sutured, and the animals were allowed torecover and to permit wound healing. In the same mice the sciatic nervebranches of the contra-lateral left hind leg were exposed but notlesioned (denoted herein “SNI contra-lateral”). Mice that underwentspared nerve injury are hereinafter denoted “SNI mice”.

2. Administration of CDK-Inhibiting Compounds to SNI Mice

After recovery from surgery and wound healing, SNI mice received peroral (p.o.) injections of CDK-inhibiting compounds.

30 mg/kg of a CDK inhibitor, dissolved in 400 μl of 2%Hydroxprolylcellulose; 0.25% Lactic Acid (85% solution) was administeredvia per oral application 30 min prior to von Frey measurements(mechanical allodynia). As a negative control, the same amount (400 μl)of 2% Hydroxprolylcellulose; 0.25% Lactic Acid (85% solution) vehiclewas administered by a single per oral application 30 min prior to vonFrey measurements.

Injection of inhibitor or vehicle, and subsequent measurements of pawwithdrawal threshold to mechanical stimulation in von Frey assays wereperformed at day 107 post SNI. Reflex nociceptive responses tomechanical stimulation were measured in a von Frey assay 30 min aftereach injection.

The effect of administration of CDK inhibitors to SNI mice on thedevelopment of mechanical allodynia was analyzed in a von Frey assay, asdescribed below.

3. Behavioral Testing of SNI Mice after Administration of CDK-InhibitingCompounds (von Frey Assay)

Mice that underwent SNI and subsequent administration of the compoundsof the present invention were tested for signs of mechanical allodyniapost nerve injury and post administration in a von Frey assay (Decosterdand Woolf, Pain 2000; 87:149-158). This assay determines the mechanicalthreshold upon which a stimulus, which normally is not painful, isrecognized by an animal as uncomfortable or painful. SNI ipsi and SNIcontra baselines, respectively, were established.

Mechanical thresholds of SNI mice were quantified using the up-downmethod based on Chaplan et al. (1994) and Malmberg and Basbaum (1998).

Mice were placed in plexiglass cylinders of about 9.5 cm in diameter, 14cm high with four vent holes toward the top and a plexiglass lid. Thecylinders were placed on an elevated mesh surface (7×7 mm squares).Prior to the day of testing, the mice were acclimated to the testingcylinders for 1-2 hours. On the day of testing the mice were acclimatedto the cylinders for about an hour, wherein the acclimation time dependson factors such as the strain of the mouse and the number of times theyhave been tested previously. In general, testing may begin once the miceare calm and stop exploring the new environment.

For testing mice, filaments 2.44, 2.83, 3.22, 3.61, 3, 84, 4.08, and4.31 (force range=0.04 to 2.0 g) were used. The 3, 61 mN filament wasapplied first. Said filament was gently applied to the plantar surfaceof one paw, allowed to bend, and held in position for 2-4 seconds.Whenever a positive response to the stimulus (flexion reaction) occurredthe next weaker von Frey hair was applied; whenever a negative response(no reaction) occurred the next stronger force was applied. The test wascontinued until the response to 4 more stimuli after the first change inresponse had been obtained. The highest force tested was 4.31. Thecut-off threshold was 2 g.

The series of scores (i.e, “flexion reaction” and “no reaction”) and theforce of the last filament applied were used to determine the mechanicalthreshold as described in Chaplan et al., Journal of NeuroscienceMethods. 53(1):55-63, 1994 July. The threshold determined is that towhich the animal would be expected to respond to 50% of the time. Micewere sacrificed after von Frey measurements were accomplished.

4. Effects of Administration of CDK-Inhibiting Compounds on theDevelopment of Neuropathic Pain

CDK-inhibiting compounds were administered to SNI mice as describedabove. Von Frey measurements were performed at ipsi-lateral andcontra-lateral paws of the animals at day 107 after surgery as describedabove. Without pharmacological treatment SNI mice show a stableallodynia after SNI surgery. Animals treated with compound display asignificant increase of threshold values indicating reduced sensitivityto mechanical stimuli (reduced allodynia). The observation of reducedallodynia signify that a CDK-inhibiting compound is effective as ahypoalgesic drug in models of chronic neuropathic pain.

Example 6 Formalin Assay—Model of Inflammatory Processes/Inflammatoryand Chronic Neuropathic Pain

The formalin assay in mice is a valid and reliable behavioral model ofnociception and is sensitive to various classes of analgesic drugs(Hunskaar S, Hole K, Pain. 1987 July; 30(1):103-14.) The noxiousstimulus is an injection of 10 μl diluted formalin (2% in saline)subcutaneous or intraplantar into the left hind paw. The response islicking and flinching of the injected paw. The response shows twophases, which reflect different parts of the inflammatory process(Abbott et al 1995), an early/acute phase 0-5 min post-injection, and alate/chronic phase 5-30 min post-injection. The following protocoldescribes one possible way to conduct the experiment:

1. Injection of Formalin and Administration of CDK-Inhibiting Compound

Age, sex and weight matched wildtype mice (C3HeB/FeJ) are used in thisassay. Prior to formalin injection the animals are randomly subdividedinto experimental groups of 10 animals each. Thirty minutes prior toformalin injection, a suitable dose of a CDK inhibitor dissolved in (400μl) of 2% Hydroxprolylcellulose; 0.25% Lactic Acid (85% solution)) canbe administered by i.p. injection. Similarly, Iκ Kinase (IKK) inhibitor(30 mg/kg) in (400 μl) of 2% Hydroxprolylcellulose; 0.25% Lactic Acid(85% solution) (positive control), or vehicle alone ((400 μl) of 2%Hydroxprolylcellulose; 0.25% Lactic Acid (85% solution)) (negativecontrol) can be administered by i.p. injection 30 min before formalininjection.

For formalin injection the mouse is held with a paper towel, in order toavoid disturbance of the injection by movements. The injected hind pawis held between thumb and forefinger and 10 μl of Formalin (2%) isinjected subcutaneously (s.c.) between the two front tori into theplantar hind paw using a Hamilton syringe. The behavior of the formalin-and inhibitor-treated mice is analyzed as described below.

2. Behavioral Analysis of Mice after Injection of Formalin andAdministration of CDK-Inhibiting Compound

The behaviour of the formalin-treated mice, i.e. licking and flinching,is monitored by an automated tracking system (Ethovision 3.0 Color Pro,Noldus, Wageningen, Netherlands) over a defined period of time:measurement is initiated 5 min after formalin injection and terminated30 min after formalin injection. This time frame covers phase II offormalin-induced nociception (pain), which is hyperalgesia.

Two different fluorescent dyes are used for topically marking theinjected hind paw (yellow dye) (Lumogenyellow; BASF Pigment, Cologne,Germany) and the contralateral paw (blue dye) (Lumogenviolet; KremerPigmente, Aichstetten, Germany) respectively. To determine lickingbehaviour, mice are monitored with a CCD camera. After monitoring andrecording, the video is analyzed using the EthoVision software(Ethovision 3.0 Color Pro, Noldus, Wageningen, Netherlands) or by manualanalysis. Fluorescent dot sizes and fluorescence intensities weremeasured and reduction of fluorescent dot size through licking andbiting was calculated. The overall licking time intensity wasautomatically calculated by comparison of dot size reduction of treatedversus untreated paws.

As another variant of assay read out the licking behaviour of theindividual animals was tracked manually based on video files. Lickingtimes were recorded over 30 minutes after formalin injection andsubdivided for three different licking zones (dorsum, plantar, toes).Overall licking times can be calculated for each animal as well as eachexperimental group and be used as a parameter for determination ofcompound efficacy.

As a result it was found that mice receiving vehicle treatment prior toformalin injection (negative control) displayed a prolonged licking timeand a significant reduction of fluorescent dot size at theformalin-treated paw.

In contrast, a reduction in licking time and in consequence nosignificant reduction of fluorescent dot size of the formalin-treatedpaw could be observed in test compound/formalin-treated mice. The sameeffect, i.e. a reduction in licking time and a minor change influorescent dot size, was observed in control mice treated with Ikappakinase inhibitor (IKK; for function of IKK see FIG. 2, positivecontrol).

This observation is indicative for reduced inflammatory/chronicinflammatory pain perception in CDK9 inhibitor-treated mice and for ahypoalgesic effect of the tested compound.

Example 7 Carrageenan Assay in Mice—Model of Inflammation andInflammatory Pain

The model of carrageenan induced paw edema is a standard laboratoryassay used to predict anti-inflammatory activity and reduction ofinflammation-induced pain perception of respective compounds. Thefollowing protocol describes one possible way to conduct the experiment.

The basic measurement constitutes in the measurement of edema andmechanical as well as thermal hypersensitivity in response to irritants,such as carrageenan.

Inflammation and resulting inflammatory pain is induced by subcutaneousinjection of 25 μl of 1% carrageenan (in saline) into mice hind paw(ipsi-lateral paw). Each group of 10 mice receives administration of acompound according to Formula I, 30 mg/kg body weight, vehicle ((400 μl)of 2% Hydroxprolylcellulose; 0.25% Lactic Acid (85% solution)) andsaline (physiol. NaCl) by i.p. injection 30 min prior to carrageenaninjection. Contra-lateral paws do not receive carrageenan injection.

1.1 Effects of Administration of a CDK-Inhibiting Compound onCarrageenan-Treated Mice

Paw edema induced by carrageenan injection are detected by increased pawsize measured from dorsal to plantar at the metatarsus region of theinjected (ipsi-lateral) paws. Sizes of ipsi- and contra-lateral pawsserve as surrogate markers for inflammation and are measured at severaltime points after carrageenan injection: before injection (−1), 2 h (2),3 h (3) 4 h (4), 5 h (5), 6 h (6), 24 h (24) after injection.

The paw size of all mice may increase, e.g., by 2 to 3 mm (+10%) withinthe first hour after carrageenan injection, independent of the type oftreatment substance injected 30 minutes prior to carrageenan. During thetime course, mice which received treatment with a CDK-inhibitingcompound prior to carrageenan injection may display a reduction of theedema until 24 h after carrageenan injection: the increase in paw sizecould drop e.g. from 10% down to 8%. In contrast, the paw size of thecontrol mice could increase by 30% in average at this time point. After24 h post carrageenan injection, the size of all paws treated withcarrageenan may increase to reach its maximum at 96 h after injection.

As a read-out of the carrageenan assay, a Hargreaves Assay may beperformed, wherein said assay allows the measuring of thermalsensitivity to radiant heat. The Hargreaves assay (Hargreaves et al.,1988) measures nociceptive sensitivity in a freely moving animal byfocusing a radiant heat source on the plantar surface of an animal'shindpaw as it stands in a plexiglass chamber. Specifically, the lowerside of a paw is exposed to a luminous source, generating a temperatureof, e.g. 55° C. Thermal sensitivity is measured as latency between startof exposure and lifting/pulling the exposed paw.

Mice treated with a CDK9 inhibitor as disclosed herein and carrageenan,or with Naproxen and carrageenan, or with solvent and carrageenan,respectively, are subjected to a Hargreaves assay. Mice treated with aCDK inhibitor and carrageenan could display a longer latency, comparedto negative control mice. This observation would be indicative for ahypoalgesic effect of the CDK inhibitors as disclosed herein.

Example 8 Carrageenan Assay in Rats—Model of Inflammation andInflammatory Pain

The following depicts one possible way of performing the carrageenanassay in rats.

Said assay detects analgesic/anti-inflammatory activity in rats withinflammatory pain, following the protocol as described by Winter et al(Proc. Soc. Exp. Biol. Med., 111, 544-547, 1962).

Rats (200-250 g) are injected with a suspension of carrageenan into thelower surface of the right hindpaw (0.75 mg per paw in 0.05 mlphysiological saline). Two hours later rats are submitted consecutivelyto tactile and thermal stimulation of both hindpaws.

For tactile stimulation, the animal is placed under an inverted acrylicplastic box (18×11.5×13 cm) on a grid floor. The tip of an electronicVon Frey probe (Bioseb, Model 1610) is then applied with increasingforce first to the non-inflamed and then the inflamed hindpaw and theforce required to induce paw-withdrawal is automatically recorded. Thisprocedure is carried out 3 times and the mean force per paw iscalculated.

For thermal stimulation, the apparatus (Ugo Basile, Reference: 7371)consists of individual acrylic plastic boxes (17×11×13 cm) placed uponan elevated glass floor. A rat is placed in the box and left free tohabituate for 10 minutes. A mobile infrared radiant source (96±10mW/cm²) is then focused first under the non-inflamed and then theinflamed hindpaw and the paw-withdrawal latency is automaticallyrecorded. In order to prevent tissue damage the heat source isautomatically turned off after 45 seconds.

After the behavioral measures, the paw edema is evaluated by measuringthe volume of each hindpaw using a digital plethysmometer (Letica, Model7500), which indicates water displacement (in ml) induced by pawimmersion.

10 rats are studied per group. The test is performed blind.

The test substance, such as a CDK inhibitor according to Formula I aspresented herein, will be evaluated at 2 doses (10 and 30 mg/kg),administered p.o. 60 minutes before the test, and compared with avehicle control group.

Morphine (128 mg/kg p.o.) and acetylsalicylic acid (512 mg/kg p.o.),administered under the same experimental conditions, will be used asreference substances.

The experiment will therefore include 6 groups. Data will be analyzed bycomparing treated groups with vehicle control using unpaired Student's ttests.

Rats treated with a CDK9 inhibitor as disclosed herein and carrageenan,or with Naproxen and carrageenan, or with solvent and carrageenan,respectively, are subjected to a Hargreaves assay. Rats treated with aCDK inhibitor and carrageenan should display a longer latency, comparedto negative control rats. This observation would be indicative for ahypoalgesic effect of the CDK inhibitors as disclosed herein.

Example 9 A. In Vitro Kinase Inhibition Assays

IC50 profiles of compounds 1-3 were determined for cyclin-dependentkinases CDK2/CycA, CDK4/CycD1, CDK5/p35NCK, CDK6/CycD1 and CDK9/CycT inenzymatic kinase inhibition assays in vitro. IC50 values as obtained inthese assays were used for evaluating the specific selectivity andpotency of the compounds with respect to CDK9 inhibition.

Results obtained in these assays were used to select compoundsdisplaying specificity for CDK5. Specifically, it was intended todistinguish the CDK5-specific compounds from other compounds havingsignificant inhibitory potency also with regard to other CDKs, i.e. onsome or all of CDKs 2, 4, 6, and 9. This separation is essential inorder to avoid adverse (cytostatic/cytotoxic) effects, which may occurupon inhibition of cell cycle relevant CDKs 2, 4, 6, and 9.

Furthermore, these data were used to establish structure activityrelationships (SAR) supporting the design of new and even improvedstructures/compounds with respect to potency and selectivity.

1. Test compounds Compounds were used as 1×10⁻⁰² M stock solutions in100% DMSO, 100 μl each in column 2 of three 96-well V-shapedmicrotiterplates (in the following, said plates are referred to as“master plates”).

Subsequently, the 1×10⁻⁰² M stock solutions in column 2 of the masterplates were subjected to a serial, semi-logarithmic dilution using 100%DMSO as a solvent, resulting in 10 different concentrations, thedilution endpoint being 3×10⁻⁰⁷ M M/100% DMSO in column 12. Column 1 and7 were filled with 100% DMSO as controls. Subsequently, 2×5 μl of eachwell of the serial diluted copy plates were aliquoted in 2 identicalsets of “compound dilution plates”, using a 96-channel pipettor.

On the day of the kinase inhibition assay, 45 μl H₂O were added to eachwell of a set of compound dilution plates. To minimize precipitation,the H₂O was added to the plates only a few minutes before the transferof the compound solutions into the assay plates. The plates were shakenthoroughly, resulting in “compound dilution plates/10% DMSO” with aconcentration of 1×10⁻⁰³ M/10% DMSO to 3×10⁻⁰⁸ M/10% DMSO in semilogsteps. These plates were used for the transfer of 5 μl compound solutioninto the “assay plates”. The compound dilution plates were discarded atthe end of the working day. For the assays (see below), 5 μl solutionfrom each well of the compound dilution plates were transferred into theassay plates. The final volume of the assay was 50 μl. All compoundswere tested at 10 final assay concentrations in the range from 1×10⁻⁰⁴ Mto 3×10⁻⁰⁹ M. The final DMSO concentration in the reaction mixtures was1% in all cases.

2. Recombinant Protein Kinases

For the determination of inhibitory profiles, the following 5 proteinkinases were used: CDK2/CycA, CDK4/CycD1, CDK5/p35NCK, CDK6/CycD1 andCDK9/CycT. Said protein kinases were expressed in Sf9 insect cells ashuman recombinant GST-fusion proteins or His-tagged proteins by means ofthe baculovirus expression system. Kinases were purified by affinitychromatography using either GSH-agarose (Sigma) or Ni-NTH-agarose(Qiagen). The purity of each kinase was determined by SDS-PAGE/silverstaining and the identity of each kinase was verified by western blotanalysis with kinase specific antibodies or by mass spectroscopy.

3. Protein Kinase Assay

All kinase assays were performed in 96-well FlashPlates™ from PerkinElmer/NEN (Boston, Mass., USA) in a 50 μl reaction volume. The reactionmixture was pipetted in four steps in the following order:

-   -   20 μl of assay buffer (standard buffer)    -   5 μl of ATP solution (in H₂O)    -   5 μl of test compound (in 10% DMSO)    -   10 μl of substrate/10 μl of enzyme solution (premixed)

The assay for all enzymes contained 60 mM HEPES-NaOH, pH 7.5, 3 mMMgCl₂, 3 mM MnCl₂, 3 μM Na-Orthovanadate, 1.2 mM DTT, 50 μg/ml PEG20000,1 μM [□-³³P]-ATP (approx. 5×1005 cpm per well).

The following amounts of enzyme and substrate were used per well:

Kinase Kinase Substrate # Kinase Lot # ng/50 μl Substrate ng/50 μl 1CDK2/CycA SP005 100 Histone H1 250 2 CDK4/CycD1 SP005 50 Rb-CTF (Lot009) 500  3. CDK5/ SP001 50 Rb-CTF (Lot 009) 1000 p35NCK 3 CDK6/CycD1SP003 400 Rb-CTF (Lot 009) 500 4 CDK9/CycT 003 100 Rb-CTF (Lot 009) 1000

Reaction mixtures were incubated at 30° C. for 80 minutes. The reactionwas stopped with 50 μl of 2% (v/v) H₃PO₄, plates were aspirated andwashed two times with 200 μl H₂O or 200 μl 0.9% (w/v) NaCl.Incorporation of ³³P was determined with a microplate scintillationcounter (Microbeta, Wallac).

All assays were performed with a BeckmanCoulter/Sagian robotic system.

4. Evaluation of Raw Data

The median value of the counts in column 1 (n=8) of each assay plate wasdefined as “low control”. This value reflects unspecific binding ofradioactivity to the plate in the absence of a protein kinase but in thepresence of the substrate. The median value of the counts in column 7 ofeach assay plate (n=8) was taken as the “high control”, i.e. fullactivity in the absence of any inhibitor. The difference between highand low control was referred to as 100% activity. As part of the dataevaluation, the low control value from a particular plate was subtractedfrom the high control value as well as from all 80 “compound values” ofthe corresponding plate. The residual activity (in %) for each well of aparticular plate was calculated by using the following formula:

Res. Activity (%)=100×[(cpm of compound−low control)/(high control−lowcontrol)]

The residual activities for each concentration and the compound IC50values were calculated using Quattro Workflow V2.0.1.3 (Quattro ResearchGmbH, Munich, Germany; www.quattro-research.com). The model used was“Sigmoidal response (variable slope)” with parameters “top” fixed at100% and “bottom” at 0%.

It turns out that the IC50 values of compounds 1-3 are all comprisedbetween 1 nM and 10 μM.

Example 10 In Vitro SH-SY5Y Assay—Using Tau Phosphorylation toCharacterize CDK5 Inhibitors

Abnormal tau phosphorylation and deregulated CDK5/p35 activity areclosely linked to each other and represent hallmarks ofneurodegenerative diseases such as Alzheimer's disease, Parkinson'sdisease or Amyotrophic lateral sclerosis.

The human neuroblastoma cell line SH-SY5Y can be utilized as an in vitromodel to investigate CDK5 function and the efficacy of CDK5 specificinhibitors. SH-SY5Y cells express tau protein which harbors variousphosphorylation sites known to be phosphorylated by several kinasesincluding CDK5. SH-SY5Y cells can be differentiated into neuron-likecells by exposure to 10 μM all trans-retinoic acid for 3 days andsubsequent exposure to 50 ng/ml human recombinant brain-derivedneurotrophic factor for 3-5 days, thereby upregulating neuronal markers,including tau protein. In addition CDK5 and p35 protein levels wereincreased during differentiation. RA-BDNF-differentiated cells cantherefore serve as a suitable model for studying tau phosphorylation andto screen potential CDK5 inhibitors (Jämsä et al., 2004. BBRC 319;993-1000).

1. Growth and Differentiation of SH-SY5Y Cells

SH-SY5Y neuroblastoma cells (ATCC, CRL-2266) are grown in DMEMsupplemented with 15% FCS and 1% Pen/Strep at 37° C. and 5% CO₂. Fordifferentiation, cells are seeded at a density of 2×10̂5 cells/6-w innormal growth medium and incubated overnight to attach. Differentiationis induced by replacing normal growth medium with antibiotic-free growthmedium supplemented with 10 μM all trans-retinoic acid (RA, dissolved inDMSO; Sigma-Aldrich, R2625) and further incubation for 3 days.Subsequently the medium is changed to serum- and antibiotic-free mediumsupplemented with 50 ng/ml human recombinant brain-derived neurotrophicfactor (BDNF, dissolved in sterile ddH₂O; Sigma-Aldrich, B3795) andcells are incubated for another 3-5 days to complete differentiation.

Alternative Assays and Readouts to Characterize CDK5 Inhibitors

Alternatively, instead of SH-SY5Y cells human IMR-32 neuronal cellsdifferentiated with 10 μM BrdU for 3 days or rat PC12 neuroblastomacells differentiated with 50 ng/ml human recombinant nerve growth factor(NGF) for 3 days can be used to study tau phosphorylation at variousphosphorylation sites.

IMR-32 neuroblastoma cells (DSMZ, ACC 165) are grown in RPMIsupplemented with 20% FCS, 1% non-essential amino acids and 1% Pen/Strepat 37° C. and 5% CO₂. For differentiation cells are seeded at a densityof 1×10̂6 cells/6-well in normal growth medium and incubated overnight toattach. Differentiation is induced by replacing normal growth mediumwith antibiotic-free growth medium supplemented with 10 μM(+)-5-Bromo-2′-deoxyuridine (BrdU, dissolved in sterile PBS; SigmaAldrich, 858811) and further incubation for 3 days.

PC12 neuroblastoma cells (DSMZ, ACC 159) are grown in RPMI1640supplemented with 10% horse serum, 5% FCS and 1% Pen/Strep in cultureflasks coated with rat tail collagen (Roche, 11 179 179 001). To inducedifferentiation to neuron-like cells, medium is changed to serum- andantibiotic-free medium supplemented with 50 ng/ml human recombinantnerve growth factor (NGF) and further incubation for 3 days.

(C2C12 myoblasts are grown in DMEM supplemented with 10% FCS and 1%Pen/Strep. For differentiation cells are seeded at a density of 1.25×10̂5cells/12-well in normal growth medium and incubated to confluency.Differentiation is induced by replacing normal growth medium with DMEMsupplemented with 2% horse serum and 1% Pen/Strep for 2-5 days.)

2. Treatment of Differentiated SH-SY5Y Cells with CDK-InhibitingCompounds

After differentiation is completed, the medium is replaced withserum-free growth medium with CDK-inhibiting compounds as well asreference compounds such as positive and negative controls, eachdissolved in DMSO, are added at concentrations ranging from 0.1 to 100μM (final concentration of DMSO in the well should be 0.1%). Cells areincubated for 90-120 min with compounds. Cells are harvested byscraping, washed twice with PBS and lysed in lysis buffer (BioSource,FNN0011). Lysed cells are stored at −20° C. or used immediately forWestern Blot or ELISA assay to determine total tau and pS396 or pT231tau.

3. Determination of Total and Ptau Contents in SH-SY5Y Cell Lysatesafter Administration of CDK-Inhibiting Compounds

Concentrations of total tau, pS396 tau and pT231 tau within the cellculture lysates are measured by using commercial ELISA Kits (BioSource:human total tau: KHB0042; human pS396 tau: KHB7031; human pT231 tau:KHB7051) according to the manufacturers instructions or by using WesternBlots probing with total and phospho-specific antibodies (antibodies:total tau: Santa Cruz, sc-21796; pS396 tau: Santa Cruz, sc-12414; pT231tau, AT180: Pierce Endogen, MN1040; pS202 tau: AT8: Pierce Endogen,MN1020).

4. Effects of Treatment with CDK-Inhibiting Compounds on thePhosphorylation of Tau Protein at Serin396 in Differentiated SH-SY5YCells

CDK5-inhibitory compounds were dissolved in DMSO and administered to RA-and BDNF-differentiated SH-SY5Y cells in duplicates or triplicates.After 90-120 min of incubation with test or reference compounds (e.g.LiCl, a GSK3β inhibitor or roscovitine, a CDK inhibitor), cells wereharvested for Western Blot or ELISA analysis as described above.

Cells treated with compound 58 displayed a significant inhibitory effectof compound 58 on Tau phosphorylation at Serin396. Compared to referencecompounds LiCl and roscovitine, this compound exhibited a similar orbetter inhibition of Tau phosphorylation at S396.

4. Alternative Assays and Readouts

Alternative assays: human SH-SY5Y cells (differentiated with RA andBDNF) human IMR-32 neuronal cells (differentiated with BrdU) and mouseC2C12 myoblasts (differentiated to myotubes with DMEM supplemented with2% horse serum and 1% Pen/Strep for 2-5 days) express the transcriptionfactor MEF2D which is a known substrate of CDK5. CDK5/p35 phosphorylatesand thereby inhibit MEF2D transcriptional activity. Inhibition of CDK5will therefore decrease MEF2D transcriptional activity. Cellular assaysreading out MEF2D activity (e.g. using a reporter gene assay, anactivity ELISA (Panomics, TransBinding MEF2 Assay kit, EK1081) or inWestern Blots using pMEF2D and total MEF2D antibodies) will be usefultools to measure CDK5 activity. Similar readouts using transcriptionalactivity of Stat3, another known substrate of CDK5/p35 can be used (Fuet al., 2004. PNAS, vol 101(17); 6728-6733).

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1. A method for treatment of a disease selected from the group of pain,an inflammatory disorder, an immunological disease, a proliferativedisease, an infectious disease, a cardiovascular disease and aneurodegenerative disease, comprising administering a therapeuticallyeffective amount of at least one compound represented by the generalFormula I

wherein R¹ is —XSO₂NR⁵R⁶ or —XSO₂R⁸; X is a branched or unbranched C₁₋₄alkylene, wherein said C₁₋₄ alkylene optionally can be bound to R⁵ or R⁶to form a 5- or 6-membered heterocycle; R⁵ and R⁶ independently of eachother are selected from the group consisting of hydrogen, C₁₋₄alkyl,hydroxy-C₁₋₄alkyl, C₃₋₄alkenyl, C₃₋₈-cycloalkyl,C₃₋₈-cycloalkyl-C₁₋₄alkyl, C₄₋₇heterocycloalkyl-C₀₋₄alkyl,C₄₋₇-aryl-C₀₋₄alkyl, and C₄₋₇heteroaryl-C₀₋₄alkyl; or wherein R⁵ and R⁶together with the N-atom to which they are bound form a 5- to 8-memberedheterocycloalkyl, wherein said cycloalkyl, heterocycloalkyl, aryl,heteroaryl or alkyl is further optionally substituted by up to 2radicals selected from the group consisting of halo, hydroxy,aminocarbonyl, C₁₋₄ alkyl, hydroxy-C₁₋₄ alkyl, C₁₋₄ alkyl-O—C₁₋₄ alkyl,C₁₋₄ alkyl-O—, and —NR⁵R⁶; R⁸ is C₁₋₄ alkyl, hydroxy-C₂₋₄alkyl,C₃₋₄alkenyl, C₃₋₈-cycloalkyl, C₃₋₈-cycloalkyl-C₁₋₄alkyl, orC₄₋₇heterocycloalkyl-C₀₋₄ alkyl; wherein said cycloalkyl,heterocycloalkyl or alkyl is further optionally substituted by up to 2radicals selected from the group consisting of halo, hydroxy, C₁₋₄alkyl, hydroxy-C₁₋₄ alkyl, C₁₋₄ alkyl-O—C₁₋₄ alkyl, C₁₋₄ alkyl-O, and—NR⁵R⁶; R² is one or two substituents independently selected fromhalogen and hydrogen; R³ can be 1 to 3 substituents each independentlyselected from the group consisting of hydrogen, halo, hydroxy,C₁₋₄alkyl, C₃₋₇cycloalkyl, C₁₋₄ alkyl-cycloalkyl, C₁₋₄alkyl-heterocycloalkyl, —O-heterocycloalkyl, C₁₋₄ alkoxy, C₂₋₄alkenyloxy, —OCF₃, C₂₋₄ alkanoyl, C₁₋₄alkylsulfonyl, mono- anddi-(C₁-C₄alkyl)sulfonamido, aminocarbonyl, mono- anddi-(C₁-C₄alkyl)aminocarbonyl, aryl-C₁₋₄ alkoxy, heteroaryl-C₁₋₄ alkoxy,heterocycloalkyl-C₁₋₄-alkoxy, heterocycloalkyl-C₁₋₄-alkyl,heteroaryl-C₁₋₄-alkyl, C₁₋₄ alkyloxymethyl, hydroxy-C₁₋₄alkyloxymethyl,cyano, —COOH, and C₁-C₄ alkoxycarbonyl, wherein the above mentionedsubstituents can be further substituted by radicals selected from thegroup consisting of C₁₋₄-alkyl, hydroxyl-C₀₋₄-alkyl, C₁₋₄-alkoxy,aminocarbonyl, halo, and NR⁵R⁶; R^(4a) and R^(4b) are the same ordifferent and each is independently hydrogen, C₁₋₄ alkyl, or —NR′R″,wherein R′ and R″ are each independently hydrogen or C₁₋₄alkyl; and theN-oxide derivatives, prodrug derivatives, protected derivatives,individual isomers and mixtures of isomers thereof; and thepharmaceutically acceptable salts, solvates, and hydrates of suchcompounds to a patient suffering from said disease.
 2. The method ofclaim 1, wherein the pain comprises chronic pain, inflammatory pain,neuropathic pain, or a combination thereof.
 3. A method for treatment ofa disease selected from the group of pain, an inflammatory disorder, animmunological disease, a proliferative disease, an infectious disease, acardiovascular disease and a neurodegenerative disease, comprisingadministering a therapeutically effective amount of at least onecompound represented by general Formula I wherein R³ is 1 to 3substituents independently selected from the group consisting of methyl,ethyl, hydroxymethyl, hydroxy, methoxy, ethoxy, isopropoxy, benzyloxy,hydrogen, fluoro, chloro, trifluoromethyl, 2-methoxy-ethoxy,methoxymethyl, 2-methoxy-ethyl, tetrahydro-furan-3-yloxy,tetrahydro-furan-2-yl-methoxy, —N(CH₃)SO₂CH₃, piperidin-1-yl-methyl,2-hydroxymethyl-piperidin-1-yl-methyl,3-hydroxymethyl-piperidin-1-yl-methyl,3-(2-hydroxy-ethyl)-piperidin-1-yl-methyl,3-aminocarbonyl-piperidin-1-yl-methyl, dimethylaminomethyl,diethylaminomethyl, (ethyl-isopropyl-amino)-methyl,morpholin-4-ylmethyl, 4-methyl-piperazin-1-yl-methyl,[1,2,4]triazol-1-yl-methyl, pyridine-3-yl-methoxy, andpyridine-4-yl-methoxy to a patient suffering from said disease.
 4. Themethod of claim 3, wherein the pain comprises chronic pain, inflammatorypain, neuropathic pain, or a combination thereof.
 5. A method fortreatment of a disease selected from the group of pain, an inflammatorydisorder, an immunological disease, a proliferative disease, aninfectious disease, a cardiovascular disease and a neurodegenerativedisease, comprising administering a therapeutically effective amount ofat least one compound represented by general Formula Ia

wherein R¹ is —CH₂SO₂NR⁵R⁶ or —CH₂SO₂R⁸, R² is hydrogen, R^(3a) ishydrogen or C₁₋₄ alkoxy, R^(3b) is hydrogen, C₁₋₄ alkyl optionallysubstituted by dialkylamine, heterocycloalkyl-C₁₋₄-alkyl optionallysubstituted by C₁₋₄-alkyl or by hydroxyl-C₀₋₄-alkyl, orheteroaryl-C₁₋₄-alkyl; R^(3c) is hydrogen or halogen, R^(4a) and R^(4b)are each independently C₁₋₄ alkyl or hydrogen, R⁵ is selected from thegroup consisting of hydrogen, methyl, 2-hydroxyethyl, cyclobutyl,cyclopentyl, 2-dimethylaminoethyl, 3-dimethylaminopropyl,tetrahydro-furan-3-yl, pyrrolidin-3-yl, pyridine-3-yl, pyridin-4-yl, and4-piperidinyl; R⁶ is hydrogen or methyl, R⁸ is hydroxy-C₂₋₄-alkyl, andthe N-oxide derivatives, prodrug derivatives, protected derivatives,individual isomers and mixtures of isomers thereof; and thepharmaceutically acceptable salts, solvates, and hydrates of suchcompounds to a patient suffering from said disease.
 6. The method ofclaim 5, wherein the pain comprises chronic pain, inflammatory pain,neuropathic pain, or a combination thereof.
 7. A method for treatment ofa disease selected from the group of pain, an inflammatory disorder, animmunological disease, a proliferative disease, an infectious disease, acardiovascular disease and a neurodegenerative disease, comprisingadministering a therapeutically effective amount of at least onecompound selected from the group consisting of:{4-[4-(2-Methoxy-phenyl)-pyrimidin-2-ylamino]-phenyl}-methanesulfonamide(Compound 1);C-{4-[4-(2-Methoxy-phenyl)-pyrimidin-2-ylamino]-phenyl}-N-methyl-methanesulfonamide(Compound 2); and{4-[4-(2-Methoxy-phenyl)-6-methyl-pyrimidin-2-ylamino]-phenyl}-methanesulfonamide(Compound 3) to a patient suffering from said disease.
 8. The method ofclaim 7, wherein the pain comprises chronic pain, inflammatory pain,neuropathic pain, or a combination thereof.